MEDICAL 


Gift  of 
Hahnemann  Medical  College 


A  TEXT-BOOK 


OF 


BACTERIOLOGY 


BY 

GEORGE  M.  STERNBERG,  M.D.,  LL.D. 

SURGEOX-GEXERAL    U.    S.    ARMY 

EX-PRESIDENT  OF  THE  AMERICAN  MEDICAL  ASSOCIATION  AND  OF  THE  AMERICAN  PUBLIC  HEALTH 

ASSOCIATION;  HONORARY  MEMBER  OF  THE  EPIDEMIOLOGICAL  SOCIETY  OF  LONDON,  OP 

THE  ROYAL  ACADEMY  OF  MEDICINE  OF  ROME,  OF  THE  ACADEMY  OF  MEDICINE  OF 

RIO  DE  JANEIRO,  OF  THE  SOCI^Tfi  FRAN9AISE  D'HYGIENE,  ETC.,  ETC. 


ILLUSTRATED  BY  HELIOTYPE  AND  CHROMO-LITHOGRAPHIC  PLATES 
AND  TWO  HUNDRED  ENGRAVINGS. 


Seconb  IRevteet)  Edition 


NEW   YOEK 
WILLIAM   WOOD   AND  COMPANY 

MDCCCCI 


COPYRIGHT  BY 
WILLIAM    WOOD   &   COMPANT, 

1901. 


PRESS  OF 

THE    PUBLISHERS'   PRINTING   COM°ANV 

32-34   LAFAYETTE   PLACE 

NEW    YORK 


•  -V 

•  •  •  I 


PREFACE. 


writer's  Manual  of  Bacteriology,  published  in  1892,  has  been 
very  favorably  received  both  in  this  country  and  abroad,  but 
its  usefulness  has  no  doubt  been  to  some  extent  restricted  by  the 
size  and  expense  of  the  volume.  The  following  is  an  extract  from 
the  preface  of  the  Manual : 

"A  Manual  of  Bacteriology,  therefore,  which  fairly  represents  the 
present  state  of  knowledge,  will  consist  largely  of  a  statement  of  facts 
established  by  experimental  data,  and  cannot  fail  to  be  of  value  to 
physicians  and  to  advanced  students  of  bacteriology  as  a  work  of 
reference.  The  present  volume  is  an  attempt  to  supply  such  a  man- 
ual, and  at  the  same  time  a  text-book  of  bacteriology  for  students 
and  guide  for  laboratory  work.  That  portion  of  the  book  which  is 
printed  in  large  type  will,  it  is  hoped,  be  found  to  give  an  accurate 
and  sufficiently  extended  account  of  the  most  important  pathogenic 
bacteria,  and  of  bacteriological  technology,  to  serve  as  a  text-book  for 
medical  students  and  others  interested  in  this  department  of  science. 
The  descriptions  of  non-pathogenic  bacteria,  and  of  the  less  important 
or  imperfectly  described  species  of  pathogenic  bacteria,  are  given  in 
smaller  type." 

For  the  benefit  of  students  of  medicine  and  others  who  do  not  care 
especially  for  the  detailed  descriptions  of  non-pathogenic  bacteria  and 
the  extensive  bibliography  contained  in  the  Manual,  this  TEXT-BOOK 
OF  BACTERIOLOGY  is  now  published.  It  comprises  that  portion  of 
the  Manual  above  referred  to  as  printed  in  large  type,  revised  to  in- 
clude all  important  additions  to  our  knowledge  of  the  pathogenic 
bacteria  since  the  original  date  of  publication. 

1896. 


2.01*78 


PREFACE  TO  SECOND  EDITION. 


A    T  the  request  of  the  publishers  the  author  has  again  under>taken 

a  revision  of  his  Manual  of  Bacteriology,  published  in  1892: 

This  is  practically  a  third  edition  of  that  work,  although  the  title 

was  changed  in   1896,  and  it  now  appears  as  a  second  edition  of  a 

TEXT-BOOK  OF  BACTERIOLOGY. 

Considerable  additions  have  been  made  to  the  present  edition,  in- 
cluding a  section  on  "  Protective  Inoculations  in  Infectious  Diseases," 
and  one  on  the  "  Bacteria  of  Plant  Diseases."  In  order  that  the  size 
of  the  work  might  not  be  materially  increased,  descriptions  of  species 
imperfectly  described,  or  of  minor  importance,  have  been  omitted. 
In  the  Manual  of  Bacteriology  an  attempt  was  made  to  include  all 
species  or  distinct  varieties  which  had  been  described  by  competent 
bacteriologists  up  to  that  date,  and  to  give  a  very  full  bibliography  of 
the  subject.  It  was  found  to  be  impracticable  to  follow  this  plan  in 
bringing  out  a  second  edition  as  it  would  have  called  for  two  large 
volumes  instead  of  one,  and  the  limited  demand  for  such  a  work 
would  probably  have  made  it  a  losing  venture  for  the  publishers. 
In  the  TEXT-BOOK,  therefore,  the  bibliography  and  the  descriptions 
of  many  non-pathogenic  species  were  omitted.  The  Manual  is  now. 
out  of  print,  and  those  who  have  use  for  a  comprehensive  work,  in 
which  an  attempt  has  been  made  to  include  all  species  described  up 
to  date  of  publication,  are  referred  to  Migula's  "  System  der  Bak- 
terien  "  (Gustav  Fischer,  Jena,  1900). 
WASHINGTON,  May  27th,  1901. 


TABLE    OF    CONTEXTS. 


PART  FIRST. 

CLASSIFICATION,  MORPHOLOGY,  AND   GENERAL  BACTERIOLOGICAL 

TECHNOLOGY. 

PAGE 

I.  HISTORICAL, .               .3 

II.  CLASSIFICATION, .        .                .10 

III.  MORPHOLOGY, .                       .20 

IV.  STAINING  METHODS,       ....  ...    25 

V.  CULTURP:  MEDIA, .                        .    37 

VI.  STERILIZATION  OF  CULTURE  MEDIA, 52 

VII.  CULTURES  IN  LIQUID  MEDIA, 62 

VIII.  CULTURES  IN  SOLID  MET>IA,          ...  ...    69 

IX.  CULTIVATION  OF  ANAEROBIC  BACTERIA, 80 

X.  INCUBATING  OVENS  AND  THERMO-REGULATORS,          .        .        .88 
XI.  EXPERIMENTS  UPON  ANIMALS,      ...  ...    96 

XII.  PHOTOGRAPHING  BACTERIA,          ...  .  103 


PART   SECOND. 
GENERAL   BIOLOGICAL   CHARACTERS. 

I.  STRUCTURE,  MOTIONS,  REPRODUCTION 117 

II.  CONDITIONS  OF  GROWTH, 125 

III.  MODIFICATIONS  OF  BIOLOGICAL  CHARACTERS,     ....  129 

IV.  PRODUCTS  OF  VITAL  ACTIVITY, 133 

V.  PTOMAINES  AND  TOXALBUMINS 146 

VI.  INFLUENCE  OF  PHYSICAL  AGENTS, 153 

VII.  ANTISEPTICS  AND  DISINFECTANTS  (GENERAL  ACCOUNT  OF  THE 

ACTION  OF), 164 

VIII.  ACTION  OF  GASES  AND  OF  THE  HALOID  ELEMENTS  UPON  BAC- 
TERIA,           172 

IX.  ACTION  OF  ACIDS  AND  ALKALIES, 180 

X.  ACTION  OF  VARIOUS  SALTS, 186 

XI.  ACTION  OF  COAL-TAR  PRODUCTS,  ESSENTIAL  OILS,  ETC.,          .  197 

XII.  ACTION  OF  BLOOD  SERUM  AND  OTHER  ORGANIC  LIQUIDS,        .  208 

XIII.  PRACTICAL  DIRECTIONS  FOR  DISINFECTION,         ....  214 


VI  TABLE   OF   CONTENTS. 

PART   THIRD. 
PATHOGENIC   BACTERIA. 

PAGE 

I.  MODES  OF  ACTION, 221 

II.  CHANNELS  OF  INFECTION,    .....  .  229 

III.  SUSCEPTIBILITY  AND  IMMUNITY, 233 

IV.  PROTECTIVE  INOCULATIONS,         .                                ...  272 
V.  PYOGENIC  BACTERIA,    . 371 

VI.  BACTERIA  IN  CROUPOUS  PNEUMONIA, 396 

VII.  PATHOGENIC  MICROCOCCI    NOT    DESCRIBED   IN   SECTIONS  V. 

AND  VI.,  410 

VIII.  THE  BACILLUS  OF  ANTHRAX, 422 

IX.  THE  BACILLUS  OF  TYPHOID  FEVER, 431 

X.  BACTERIA  IN  DIPHTHERIA, 449 

XI.  BACTERIA  IN  INFLUENZA, 463 

XII.  BACILLI  IN  CHRONIC  INFECTIOUS  DISEASES,      ....  467 

XIII.  BACILLI  WHICH  PRODUCE  SEPTICAEMIA  IN  SUSCEPTIBLE   ANI- 

MALS,     498 

XIV.  PATHOGENIC  AEROBIC  BACILLI  NOT  DESCRIBED  IN   PREVIOUS 

SECTIONS, 528 

XV.  BACTERIA  IN  PLANT  DISEASES,    .        .  ....  571 

XVI.  PATHOGENIC  ANAEROBIC  BACILLI,       .        .        .        .        .        .  578 

XVII.  PATHOGENIC  SPIRILLA,         .  590 


PART   FOURTH. 
SAPROPHYTES. 

I.  BACTERIA  IN  THE  ATR, 613 

II.  BACTERIA  IN  WATER, 626 

III.  BACTERIA  IN  THE  SOIL, 642 

IV.  BACTERIA  OF  THE  SURFACE  OF  THE  BODY  AND  OF   EXPOSED 

Mucous  MEMBRANES, 648 

V.  BACTERIA  OF  THE  STOMACH  AND  INTESTINES,    ....  658 
VI.  BACTERIA  OF  CADAVERS  AND  OF  PUTREFYING  MATERIAL  FROM 

VARIOUS  SOURCES, 664 

VII.  BACTERIA  IN  ARTICLES  OF  FOOD, 667 

INDEX.          *...,.,,  .  .673 


LIST  OF  ILLUSTRATIONS. 


FIG. 

PAGE 

1. 

Staphylococci,           .         .                  .                          .... 

21 

2. 

ZodgloBa    ............ 

21 

3. 

Ascococcus,       ...                 ..... 

21 

4. 

Streptococci,     ...                 .                 ... 

21 

5 

Tetrads,    .        .                 

22 

6. 

Packets  —  sarcina,      .......... 

22 

7. 

23 

8. 

Involution  forms,      ........ 

23 

9. 

Chains  formed  by  binary  division,    

23 

10. 

Spirilla,     ......                 ..... 

24 

11. 

Cladothrix  ...... 

24 

12. 

Fla^ella,    . 

24 

13. 

Platinum  wire  in  glass  handle,          

25 

14. 

Flask  for  drawing  off  blood  serum,          

38 

15. 

Method  of  forcing  blood  serum  into  test  tube, 

38 

16. 

Suction  pipette,        .                 ........ 

38 

17. 

Hot-  water  funnel,     .                         ...                 ... 

42 

18. 

Karlinski's  agar  filter;      

45 

20. 

Glass  dishes  for  preserving  potato  cultures,     

49 

21. 

Test  tube  for  sterilizing  potato  

49 

22. 

Shape  of  potato  for  test-tube  culture;      

49 

23. 

Hot  air  oven  

54 

24. 

Koch's  steam  sterilizer,     

55 

25. 

Koch's  steam  sterilizer,     

55 

26. 

Arnold's  steam  sterilizer,           ...                 .... 

56 

27. 

Milncke's  steam  sterilizer,         

56 

28. 

Koch's  apparatus  for  coagulating  blood  serum,       .... 

57 

29. 

Miincke  s  steam  sterilizer  and  coagulator  

58 

30. 

Pasteur  Chamberlain  filter,       

59 

31. 

Pasteur  Chamberlain  filter  without  metal  case,       .... 

60 

32. 

Modified  Pasteur-Chamberlain  filter,        ...... 

61 

33. 

Erlenmeyer  flask  

63 

34. 

Flask  used  by  Pasteur,     , 

63 

35. 

Platinum  wire  loop,          *.        

64 

36. 

Platinum  needle,       

65 

37. 

Sternberg's  bulb,      

66 

38. 

Fermentation  tube,  .         

68 

39. 

Method  of  making  stick  culture,     

69 

Vlll  LIST    OF   ILLUSTRATIONS. 

FIG.  PAGE 

40.  Sloping  surface  of  culture  medium, 70 

40A.  Growth  of  non-liquefying  bacteria  in  gelatin  stick  cultures,    .                 .  70 

41.  Growth  of  same  along  line  of  puncture,  ...                          .         .  71 

42.  Growth  of  liquefying  bacilli,    ...                          ....  72 

43.  Colonies  of  bacteria, 73 

44.  Apparatus  for  gelatin  plates .75 

45.  Esmarch  roll  tube, 76 

46.  (See  Fig.  15). 

47.  Mode  of  development  of  a  facultative  anaerobic  bacillus,       .                 .  go 
4S.  Mode  of  development  of  strict  anaerobic  in  long  stick  culture,        .         .  80 

49.  Exhausted-air  flask  for  liquid  media,  .....                 .  82 

50.  Method  of  displacing  air  with  hydrogen,          .         .  82 

51.  Salomonson's  tube,    . .  82 

5'3.  Friinkel's  method  of  cultivation,      .  .  .83 

53.  Sternberg's  method  of  cultivation,   ....                  .  83 

54.  Sternberg's  method  of  cultivation,   ...                 ....  84 

55.  Buchner's  method  of  cultivation,     .....  84 

56.  Hydrogen  generator, 85 

57.  Hydrogen  apparatus  for  plate  cultures,   .......  86 

58.  Incubating  oven, .  89 

59.  Thermo-regulator  for  gas,         .                          90 

60.  Moitessier's  pressure  regulator,          .                 .                  .         .         .         .  90 

61 .  Mica  screen  for  flame,       ...                 .                 ....  9 1 

6.'.  Koch's  device  for  cutting  off  fli  MC.                                                    .         .  91 

63.  Reichert's  thermo  regulator.     .  91 

64.  Bohr's  thermo-regulator,           .                  ...                  ...  91 

65.  Miincke's  thermo-regulator      .....                  .  92 

66.  Sternberg's  thermo-regulator,  .........  92 

67.  Gas  valve  for  the  same,    .        .                 .                         ....  92 

68.  D'Arsonval's  incubating  apparatus,          ......         0  93 

69.  Roux's  incubating  oven  and  thermo-regulator,         ....  93 

70.  Roux's  thermo-regulator, 94 

71.  Koch's  syringe, 97 

72.  Sternberg's  glass  syringe, $7 

73.  Pringle's  photomicrographic  apparatus,  .         .         .         .         .         .  108 

74.  Sternberg's  photomicrographic  apparatus  for  gas,  .....  HO 

75.  Spores  of  bacilli, 121 

76.  Method  of  germination  of  spores,     ........  122 

77.  Apparatus  for  cultivating  anaerobic  bacilli, 

78.  Bacillus  of  mouse  septicaemia  in  leucocytes  from  blood  of  mouse,          .  256 

79.  Staphylococcus  pyogenes  aureus,     ........  374 

80.  Gelatin  culture  of  Staphylococcus  pyogenes  aureus,       ....  375 

81.  Vertical  section  through  a  subcutaneous  abscess  caused  by  inoculation 

with  staphylococci  in  the  rabbit, 377 

82.  Pus  containing  streptococci, .  383 

83.  Streptococcus  of  erysipelas  in  nutrient  gelatin, 384 

84.  Section  from  margin  of  anerysipelatous  inflammation,  showing  strepto- 

cocci in  lymph  spaces, 385 

85.  Gouococci 391 

86.  Gonococcus  in  gouorrhceal  pus, 392 

87.  Gonorrhoeal  conjunctivitis,  second  day  of  sickness,         '.                          .  394 


LIST    OF    ILLUSTRATIONS.  IX 

FIG.  PAGE 

88.  Friedlander's  bacillus, 

89.  Friedlander's  bacillus;  stick  culture  in  gelatin,  .  39? 

90.  Micrococcus  pneumoniae  crouposae,  ....                 .  401 

91.  Micrococcus  pneumonias  crouposae,          .        .        .  401 
9'2.  Micrococcus  pneumonias  crouposae,           .         .         .  401 

93.  Micrococcus  pneumonias  crouposae,  showing  capsule,      .         .         .  402 

94.  Single  colony  of  Micrococcus  pneumonias  crouposae  upon  agar  plate,     .  403 

95.  Micrococcus  pneumonias  crouposae  in  blood  of  rabbit  inoculated  with 

sputum,        ....                 407 

96.  Micrococcus  tetragenus, 411 

97.  Streptococcus  of  mastitis  in  cows,    .                  417 

98.  Bacillus  anthracis,  showing  development  of  long  threads  in  convoluted 

bundles, 423 

99.  Bacillus  authracis,  showing  formation  of  spores, 424 

100.  Culture  of  Bacillus  authracis  in  nutrient  gelatin,     .....  '425 

101.  Colonies  of  Bacillus  authracis  upon  gelatin  plates,  ....  426 

102.  Bacillus  anthracis  in  liver  of  mouse, 428 

103.  Bacillus  authracis  in  kidney  of  rabbit, 429 

104.  Bacillus  of  typhoid  fever;  colonies  in  stained  sections  of  spleen,     .         .  434 

105.  Bacillus  of  typhoid  fever;  colonies  in  stained  sections  of  spleen,     .         .  434 

106.  Bacillus  typhi  abdominalis, 437 

107.  Bacillus  typhi  abdominalis,       .........  437 

108.  Bacillus  typhi  abdominalis,  showing  flagella, 438 

109.  Single  colony  of  Bacillus  typhi  abdominalis  in  nutrient  gelatin,     .         .  438 

110.  Bacillus  typhi  abdominalis;  stick  culture  in  nutrient  gelatin,           .         .  439 

111.  Section  through  wall  of  intestine,  showing  invasion  by  typhoid  bacilli,  443 

112.  Bacillus  diphtherias, 453 

113.  Colonies  of  Bacillus  diphtherias  in  nutrient  agar, 454 

114.  Bacillus  tuberculosis, 469 

115.  Bacillus  tuberculosis  in  sputum, 470 

116.  Section  through  a  tuberculous  nodule  in  the  lung  of  a  cow,  showing 

two  giant  cells. 472 

117.  Tubercle  bacilli  from  surface  of  culture  upon  blood  serum,    .         .        .  475 

118.  Culture  of  tubercle  bacillus  upon  glycerin-agar, 477 

119.  Limited  epithelioid-celled  tubercle  of  the  iris, 480 

120.  Section  of  a  recent  lepra  nodule  of  the  skin, 486 

121.  Bacillus  mallei 489" 

122.  Section  of  a  glanders  nodule, 489 

123.  Section  through  a  glanders  nodule  in  liver  of  field  mouse,       .         .         .  492 

124.  Migrating  cell  containing  syphilis  bacilli, 495 

125.  Pus  from  hard  chancre  containing  syphilis  bacilli,  ....  495 

126.  Bacillus  of  rhinoscleroma  in  lymphatic  vessels  of  the  superficial  part  of 

tumor, 496 

127.  Bacillus  septicaemias  haemorrhagicae  in  blood  of  a  rabbit,        .         .        .  499 

128.  Bacillus  septicaemias  haeinorrhagicae;  stick  culture  in  nutrient  gelatin,  .  501 

129.  '  Bacillus  of  Schweineseuche, 501 

130.  Colonies  of  bacillus  of  swine  plague, 501 

131.  Bacillus  of  Schweineseuche  in  blood  of  rabbit, 502 

132.  Bacillus  of  hog  cholera,   ...  ..-.'«                 .  504 

133.  Bacillus  of  mouse  septicaemia  in  leucocytes  from  blood  of  mouse,  .        .  511 

134.  Bacillus  of  rouget 512 


X  LIST    OF   ILLUSTRATIONS. 

FIG-  PAGE 

135.  Bacillus  of  mouse  septicaemia  ;  culture  in  nutrient  gelatin,     .        .  513 

136.  Bacillus  of  mouse  septicaemia;  single  colony  in  nutrient  gelatin,     .        v  513 

137.  Section  of  diaphragm  of  a  mouse  dead  from  mouse  septicaemia,     .         ,  514 

138.  Bacillus  cavicida  Havaniensis, 51g 

139.  Bacillus  crassus  sputigenus, ,  5^7 

140.  Proteus  hominis  cnpsulatus, 5^9 

141.  Bacillus  capsulatus,           .                  523 

142.  Bacillus  hydrophilus  fuscus, 523 

143.  Culture  of  Bacillus  hydrophilus  fuscus  in  nutrient  gelatin,     .        .        ,  533 

144.  Bacillus  coli  commuuis, 530 

145.  Bacillus  coli  communis  in  nutrient  gelatin,      ....         .  533 

146.  A  portion  of  the  growth  shown  in  Fig.  147,    ......  533 

147.  Bacillus  lactis  aerogenes, 535 

148.  Bacillus  acidiformans, 537 

149.  Culture  of  Bacillus  acidiformans  in  nutrient  gelatin,       ....  537 

150.  Bacillus  cuniculicida  Havaniensis, 533 

151.  Colonies  of  Bacillus  cuniculicida  Havaniensis, 539 

152.  Colonies  of  Bacillus  cuniculicida  Havaniensis, 539 

153.  Bacillus  pyocyaneus,        ..........  543 

154.  Proteus  vulgaris 548 

155.  "  Swarming  islands  "  from  a  culture  of  Proteus  mirabilis,     .         .        .  553 

156.  Spiral  zooglcea  from  a  culture  of  Proteus  mirabilis,        ....  553 

157.  Bacillus  gracilis  cadaveris, 559 

158.  Colonies  of  Bacillus  gracilis  cadaveris, 559 

159.  Tetanus  bacillus, 579 

160.  Tetanus  bacillus 579 

161.  Culture  of  Bacillus  tetani  in  nutrient  gelatin,          .        .         .        .        »  580 
163.  Bacillus  oedematis  maligni, 581 

163.  Bacillus  oedematis  maligni, ,  581 

164.  Cultures  of  Bacillus  oedematis  maligni  in  nutrient  gelatin,      .         .        .  582 

165.  Bacillus  cadaveris, 584 

166.  Bacillus  cadaveris 584 

167.  Bacillus  of  symptomatic  anthrax, 585 

168.  Bacillus  of  symptomatic  anthrax, 585 

169.  Culture  of  bacillus  of  symptomatic  anthrax, 586 

170.  Spirillum  Obermeieri, 591 

171.  Spirillum  Obermeieri, 591 

172.  Spirillum  cholerae  Asiaticae .  593 

173.  Spirillum  cholerae  Asiaticae, 593 

174.  Colonies  of  Spirillum  cholerae  Asiaticae 594 

175.  Spirillum  cholerae  Asiaticae, 594 

176.  Cultures  of  Spirillum  cholerae  Asiaticae  in  nutrient  gelatin,     .         .        .  595 

177.  Spirillum  choleras  Asiaticae, 595 

178.  Colonies  in  nutrient  gelatin  of  Spirillum  cholene  Asiaticae,  Spirillum 

tyrogenum,  and  Spirillum  of  Finkler  and  Prior,         ....  596 

179.  Section  through  mucous  membrane  of  intestine  from  cholera  cadaver,  599 

180.  Spirillum  of  Finkler  and  Prior, 602 

181.  Colonies  of  Spirillum  of  Finkler  and  Prior, 602 

182.  Spirillum  of  Finkler  and  Prior  ;  culture  in  gelatin,         ....  602 

183.  Spirillum  tyrogenum, .  603 

184.  Colonies  of  Spirillum  tyrogenum,    .                        .....  604 


LIST   OF   ILLUSTRATIONS.  XI 

FIG.  PAGE 

185.  Spirillum  Metsclmikovi,   .  604 

186.  Penicillum  ghiucum,        ...                                                    •  614 

187.  Miquel's  aeroscope,  ....                                  .                 .  615 

188.  Hesse's  aeroscope •  617 

189.  Miquel's  flask, 619 

190.  Straus  and  Wurtz's  soluble  filter, 619 

191.  Petri's  sand  filter,      '. 620 

192.  Sugar  filter, 621 

193.  Sedgwick  and  Tuckers  apparatus, 621 

194.  Sternberg's  vacuum  tube, .  627 

195.  Lepsius'  apparatus  for  collecting  water  at  various  depths,       .         .        .  628 

196.  Koch's  plate  method, ...  629 

197.  Smear  preparation  from  liver  of  yellow-fever  cadaver,    .                          .  665 

198.  Bacillus  cadaveris  grandis, .                 .  665 


-PART    FIRST. 


CLASSIFICATION,    MORPHOLOGY,  AND  GENERAL 
BACTERIOLOGICAL  TECHNOLOGY. 

I.  HISTORICAL.    II.   CLASSIFICATION.    III.   MORPHOLOGY.     IV.  STAINING 
METHODS.    V.   CULTURE  MEDIA.     VI.   STERILIZATION  OF  CULTURE 
MEDIA.    VII.   CULTURES  IN  LIQUID  MEDIA.    VIII.  CULTURES 
IN  SOLID  MEDIA.    IX.  CULTIVATION  OF  ANAEROBIC  BAC- 
TERIA.   X.  INCUBATING  OVENS  AND  THERMO  REGU- 
LATORS.    XI.  EXPERIMENTS  UPON  ANIMALS. 
XII.    PHOTOGRAPHING  BACTERIA. 


PART    FIRST. 


I. 

HISTORICAL. 

IT  is  probable  that  Leeuwenhoeck,  "  the  father  of  microscopy/' 
observed  some  of  the  larger  species  of  bacteria  in  faeces,  putrid  in- 
fusions, etc.,  which  he  examined  with  his  magnifying  glasses  (1675), 
but  it  was  nearly  a  century  later  before  an  attempt  was  made  to  de- 
fine the  characters  of  these  minute  organisms  and  to  classify  them 
(0.  F.  Muller,  1773). 

In  the  absence  of  any  reliable  methods  for  obtaining  pure  cultures, 
it  is  not  surprising  that  the  earlier  botanists,  in  their  efforts  to  classify 
microorganisms,  fell  into  serious  errors,  one  of  which  was  to  include 
under  the  name  of  infusoria  various  motile  bacteria.  These  are  now 
generally  recognized  as  vegetable  organisms,  while  the  Infusoria  are 
unicellular  animal  organisms. 

Ehrenberg  (1838),  under  the  general  name  of  Vibrioniens,  de- 
scribes four  genera  of  filamentous  bacteria,  as  follows  : 

1.  Bacterium — filaments  linear  and  inflexible  ;  three  species. 

2.  Vibrio — filaments  linear,  snake-like,  flexible  ;  nine  species. 

3.  Spirillum — filaments  spiral,  inflexible  ;  three  species. 

4.  Spirochcete — filaments  spiral,  flexible  ;  one  species. 

These  vibrioniens  were  described  by  Ehrenberg  as  "  filiform  ani- 
mals, distinctly  or  apparently  polygastric,  naked,  without  external 
organs,  with  the  body  uniform  and  united  in  chains  or  in  filiform 
series  as  a  result  of  incomplete  division/' 

Dujajdin  (1841)  also  placed  the  vibrioniens  of  Ehrenberg  among 
the  infusoria,  describing  them  as  "  filiform  animals,  extremely  slen- 
der, without  appreciable  organization,  and  without  visible  locomotive 
organs." 

Charles  Robin  (1853)  suggested  the  relationship  of  Ehrenberg's 
vibrioniens  with  the  genus  Leptothrix,  which  belongs  to  the  algae ; 
and  Davaine  (1859)  insisted  that  the  vibrioniens  are  vegetable  organ- 


4  HISTORICAL. 

isms,  nearly  allied  to  the  algae.  His  classification  will  be  found 
in  the  "  Dictionnaire  Eiicyclop.  des  Sciences  Medicales,"  art.  "  Bac- 
teries"  (18G8).  This  view  is  also  sustained  by  the  German  botanist 
Colin  and  is  now  generally  accepted. 

Spallaiizani,  in  1776,  endeavored  to  show  by  experiment  that  the 
generally  received  theory  of  the  spontaneous  generation  of  micro- 
organisms in  organic  liquids  was  not  true.  This  he  did  by  boiling 
putrescible  liquids  in  carefully  sealed  flasks.  The  experiment  was 
not  always  successful,  but  in  a  certain  number  of  instances  the 
liquids  were  sterilized  and  remained  unchanged  for  an  indefinite 
period.  The  objection  was  raised  to  these  experiments  that  the  oxy- 
gen of  the  air  was  excluded  by  hermetically  sealing  the  flasks,  and 
it  was  claimed,  in  accordance  with  the  views  of  Gay-Lussac,  that 
free  admission  of  this  gas  was  essential  for  the  development  of  fer- 
mentation. 

This  objection  was  met  by  Franz  Schulze  (1836),  who  admitted  air 
to  boiled  putrescible  liquids  by  drawing  it  through  strong  sulphuric 
acid,  in  which  suspended  microorganisms  were  destroyed.  He  thus 
demonstrated  that  boiled  solutions,  which,  when  exposed  to  the  air 
without  any  precautions,  quickly  fell  into  putrefaction,  remained  un- 
changed when  freely  supplied  with  air  which  had  been  passed  through 
an  agent  capable  of  quickly  destroying  all  living  organisms  con- 
tained in  it. 

Schwann  (1839)  demonstrated  the  same  fact  by  another  method. 
Air  was  freely  admitted  to  his  boiled  liquids  through  a  tube  which 
was  heated  to  a  point  which  insured  the  destruction  of  suspended 
microorganisms.  The  same  author  is  entitled  to  the  credit  of  hav- 
ing first  clearly  stated  the  essential  relation  of  the  yeast  plant— 
SaccJiaromyces  cerevisice — to  the  process  of  fermentation  in  saccha- 
rine liquids,  which  results  in  the  formation  of  alcohol  and  carbonic 
acid. 

Helmholtz,  in  1843,  repeated  the  experiments  of  Schwann  with 
calcined  air,  and  arrived  at  similar  results — i.  e. ,  he  found  that  the 
free  admission  of  calcined  air  to  boiled  organic  infusions  did  not  pro- 
duce fermentation  of  any  kind. 

It  was  objected  to  these  experiments  that  the  air,  having  been 
subjected  to  a  high  temperature,  had  perhaps  undergone  some  chem- 
ical change  which  prevented  it  from  inaugurating  processes  of  fer- 
mentation. 

This  objection  was  met  by  Schroder  and  Von  Dusch  (1854)  by  a 
very  simple  device  which  has  since  proved  to  be  of  inestimable  value 
in  bacteriological  researches.  These  observers  showed  that  a  loose 
plug  of  cotton,  through  which  free  communication  with  the  external 
air  is  maintained,  excludes  all  suspended  microorganisms,  and  that 


HISTORICAL.  5 

air  passed  through  such  a  filter  does  not  cause  the  fermentation  of 
boiled  organic  liquids. 

The  experiments  of  Pasteur  and  of  Hoffman,  made  a  few  years 
later,  showed  that  even  without  a  cotton  filter,  when  the  neck  of  the 
flask  containing  the  boiled  liquid  is  long  drawn  out  and  turned  down- 
ward, the  contents  may  be  preserved  indefinitely  without  change. 
In  this  case  suspended  particles  do  not  reach  the  interior  of  the  flask, 
as  there  is  no  current  of  air  to  carry  them  upward  through  its  long- 
drawn-out  neck,  and  they  are  prevented  by  the  force  of  gravity  from 
ascending. 

Tyndall  showed  at  a  later  date  that  in  a  closed  chamber,  in  which 
the  air  is  not  disturbed  by  currents,  all  suspended  particles  settle  to 
the  floor  of  the  chamber,  leaving  the  air  optically  pure,  as  is  proved 
by  passing  a  beam  of  light  through  such  a  chamber. 

Notwithstanding  the  fact  that  the  experimenters  mentioned  had 
succeeded  in  keeping  boiled  organic  liquids  sterile  in  flasks  to  which 
the  oxygen  of  the  air  had  free  access,  the  question  of  the  possibility 
of  spontaneous  generation — heterogetiesis — still  remained  unsettled, 
inasmuch  as  occasionally  a  development  of  bacterial  organisms  did 
occur  in  such  boiled  liquids. 

This  fact  was  explained  by  Pasteur  (1860),  who  showed  that  the 
generally  received  idea  that  the  temperature  of  boiling  water  must 
destroy  all  living  organisms  was  a  mistaken  one,  and  that,  especially 
in  alkaline  liquids,  a  higher  temperature  was  required  to  insure  ster- 
ilization. His  experiments  showed  that  a  temperature  of  110°  to 
112°  C.  (230°  to  233.6°  F.),  which  he  obtained  by  boiling  under  a 
pressure  of  one  and  a  half  atmospheres,  was  sufficient  in  every  case. 
These  experiments,  which  have  been  repeated  by  numerous  investi- 
gators since,  settled  the  spontaneous-generation  controversy  ;  and  it 
is  now  generally  admitted  that  no  development  of  microorganisms 
occurs  in  organic  liquids,  and  no  processes  of  putrefaction  or  fermen- 
tation occur  in  such  liquids,  when  they  are  completely  sterilized  and 
guarded  against  the  entrance  of  living  germs  from  without. 

Pasteur  at  a  later  date  (1865)  showed  that  the  atmospheric  or- 
ganisms which  resist  the  boiling  temperature  are  in  fact  reproduc- 
tive bodies,  or  spores,  which  he  described  under  the  name  of  "  corpus- 
cles ovoides  "or  "  corpuscles  brill  ants."  Spores  had  been  previously 
seen  by  Perty  (1852)  and  Robin  (1853),  but  it  was  not  until  1876  that 
the  development  of  these  reproductive  bodies  was  studied  with  care 
by  Cohn  and  by  Koch.  The  last-named  observer  determined  the 
conditions  under  which  spores  are  formed  by  the  anthrax  bacillus. 
Five  years  later  (1881)  Koch  published  his  valuable  researches  relat- 
ing to  the  resisting  power  of  anthrax  spores  to  heat  and  to  chemical 
agents. 


6  HISTORICAL. 

The  development  of  our  knowledge  relating  to  the  bacteria, 
stimulated  by  the  controversy  relating  to  spontaneous  generation 
and  by  the  demonstration  that  various  processes  of  fermentation 
and  putrefaction  are  due  to  microorganisms  of  this  class,  has 
depended  largely  upon  improvements  in  methods  of  research. 
Among  the  most  important  points  in  the  development  of  bacterio- 
logical technique  we  may  mention,  first,  the  use  of  a  cotton  air 
filter  (Schroder  and  Von  Dusch,  1854)  ;  second,  the  sterilization  of 
culture  fluids  by  heat  (methods  perfected  by  Pasteur,  Koch,  and 
others)  ;  third,  the  use  of  the  aniline  dyes  as  staining  agents  (first 
recommended  by  Weigert  in  1877)  ;  fourth,  the  introduction  of 
solid  culture  media,  and  the  ' '  plate  method  "  for  obtaining  pure  cul- 
tures, by  Koch  in  1881. 

The  various  improvements  in  methods  of  research,  and  espe- 
cially the  introduction  of  solid  culture  media  and  Koch's  "plate 
method"  for  isolating  bacteria  from  mixed  cultures,  have  placed 
bacteriology  upon  a  scientific  basis,  and  have  shown  that  many  of 
the  observations  and  inferences  of  the  earlier  investigators  were 
erroneous  owing  to  the  imperfection  of  the  methods  employed. 

Since  it  has  been  demonstrated  that  certain  infectious  diseases  of 
man  and  the  lower  animals  are  due  to  organisms  of  this  class,  phy- 
sicians have  been  especially  interested  in  bacteriological  researches, 
and  the  progress  made  during  the  past  fifteen  years  has  been  largely 
due  to  their  investigations.  It  was  a  distinguished  French  physi- 
cian, Davaine,  who  first  demonstrated  the  etiological  relation  of  a 
microorganism  of  this  class  to  a  specific  infectious  disease.  The  an- 
thrax bacillus  had  been  seen  in  the  blood  of  animals  dying  from  this 
disease  by  Pollender  in  1849  and  by  Davaine  in  1850,  but  it  was  sev- 
eral years  later  (18G3)  before  the  last-named  observer  claimed  to 
have  demonstrated  by  inoculation  experiments  the  causal  relation  of 
the  bacillus  to  the  disease  in  question. 

The  experiments  of  Davaine  were  not  generally  accepted  as  con- 
clusive, because  in  inoculating  an  animal  with  blood  containing  the 
bacillus,  from  an  infected  animal  which  had  succumbed  to  the 
disease,  the  living  microorganism  was  associated  with  material 
from  the  body  of  the  diseased  animal.  This  objection  was  subse- 
quently removed  by  the  experiments  of  Pasteur,  Koch,  and  many 
others  with  pure  cultures  of  the  bacillus,  which  were  shown  to  have 
the  same  pathogenic  effects  as  had  been  obtained  in  inoculation  ex- 
periments with  the  blood  of  an  infected  animal. 

The  next  demonstration  of  the  causal  relation  of  a  parasitic  mi- 
croorganism to  an  infectious  malady  was  made  by'Pasteur,  who  de- 
voted several  years  to  the  study  of  an  infectious  disease  of  silkworms 
which  threatened  to  destroy  the  silk  industry  of  France— pebrine. 


HISTORICAL.  7 

In  1873  Obermeier,  a  German  physician,  announced  the  discov- 
ery, in  the  blood  of  patients  suffering  from  relapsing  fever,  of  a  mi- 
nute, spiral,  actively  motile  microorganism — the  Spirochcete  Ober- 
meier i — which  is  now  generally  recognized  as  the  specific  infectious 
agent  in  this  disease. 

The  very  important  work  of  Koch  upon  traumatic  infectious 
diseases  was  published  in  1878. 

In  1879  Hansen  reported  the  discovery  of  bacilli  in  the  cells  of 
leprous  tubercles,  and  subsequent  researches  have  shown  that  this 
bacillus  is  constantly  associated  with  leprosy  and  presumably  bears 
an  etiological  relation  to  the  disease. 

In  the  same  year  (1879)  Neisser  discovered  the  "  gonococcus  "  in 
gonorrhceal  pus. 

The  bacillus  of  typhoid  fever  was  first  observed  by  Eberth,  and 
independently  by  Koch,  in  1880,  but  it  was  not  until  1884  that  Gaff- 
ky's  important  researches  relating  to  this  bacillus  were  published. 

In  1880  Pasteur  published  his  memoir  upon  fowl  cholera,  and  the 
same  year  appeared  several  important  communications  from  this 
pioneer  in  bacteriological  research  upon  the  "  attenuation "  of  the 
virus  of  anthrax  and  of  fowl  cholera  and  upon  protective  inocula- 
tions in  these  diseases. 

In  1880  the  present  writer  discovered  a  pathogenic  micrococcus, 
which  he  subsequently  named  Micrococcus  Pasteuri,  and  which  is 
now  generally  recognized  as  the  usual  agent  in  the  production  of 
acute  croupous  pneumonia — commonly  spoken  of  as  the  "  diplococ- 
cus  pneumonise,"  but  described  in  the  present  volume  under  the 
name  of  Micrococcus  pneumonice  crouposce. 

In  1881  several  important  papers  by  Koch  and  his  colleagues  ap- 
peared in  the  first  volume  of  the  "  Mittheilungen  "  published  by  the 
Imperial  Board  of  Health  of  Germany. 

The  following  year  (1882)  Koch  published  his  discovery  of  the 
tubercle  bacillus. 

The  same  year  Pasteur  published  his  researches  upon  the  disease 
of  swine,  known  in  France  as  rouget. 

The  same  investigator  (Pasteur)  also  published  in  1882  his  first 
communication  upon  the  subject  of  rabies. 

Another  important  discovery  was  made  in  1882  by  the  German 
physicians  Lofner  and  Schiitz,  viz.,  that  of  the  bacillus  of  glan- 
ders. 

Koch  published  his  discovery  of  the  cholera  spirillum — "  comma 
bacillus  "—in  1884. 

The  same  year  (1884)  Loftier  discovered  the  diphtheria  bacillus. 

Another  important  publication  during  the  same  year  was  that  of 
Rosenbach,  who,  by  the  application  of  Koch's  methods,  fixed  defi- 


8  HISTORICAL. 

nitely  the  characters  of   the  various  microorganisms  found  in  pus 
from  acute  abscesses,  etc. 

The  tetanus  bacillus  was  discovered  in  1884  by  Nicolaier,  a  stu- 
dent in  the  laboratory  of  Prof.  Fliigge,  of  Gottingen.  That  this 
bacillus  is  the  cause  of  tetanus  in  man  has  been  demonstrated  by  the 
subsequent  researches  of  numerous  investigators.  For  an  exact  knowl- 
edge of  its  biological  characters  we  are  especially  indebted  to  Kitasato. 

So  far  as  human  pathology  is  concerned,  no  important  pathogenic 
microorganism  was  discovered  after  the  year  1884  until  the  year  1892. 
After  numerous  unsuccessful  researches  by  competent  bacteriologists, 
a  bacillus  was  discovered  by  Pfeiffer,  of  Berlin,  and  independently 
by  Canon,  which  is  believed  to  be  the  specific  cause  of  influenza. 

In  1894  the  distinguished  Japanese  bacteriologist,  Kitasato,  dur- 
ing a  visit  to  China  made  for  the  purpose,  discovered  the  bacillus 
of  the  bubonic  plague  of  the  Orient. 

Finally,  we  may  refer  to  the  discovery  of  the  antitoxins  of  diph- 
theria and  of  tetanus  as  among  the  most  important  events  in  the 
history  of  bacteriology  and  of  scientific  medicine.  The  name  of  Behr- 
ing  has  the  first  place  in  connection  with  this  discovery. 

Having  briefly  passed  in  review  some  of  the  principal  events  in 
the  progress  of  our  knowledge  in  this  department  of  scientific  investi- 
gation, it  will  be  of  interest  to  students  to  know  something  more  of 
the  literature  of  bacteriology.  Important  papers  have  appeared  in 
medical  and  scientific  journals  in  all  countries,  and  research  work  of 
value  has  been  done  by  enthusiastic  investigators  of  nearly  every 
nation.  The  brilliant  pioneer  work  done  by  Pasteur  and  by  Koch  has 
attracted  to  them  many  pupils  and  has  made  France  and  Germany 
the  leading  countries  in  this  line  of  investigation.  The  very  great 
advantages  of  Koch's  methods  of  research,  introduced  at  the  com- 
mencement of  the  last  decade,  have  attracted  many  students  from 
various  parts  of  the  world  to  Berlin,  and  to  other  cities  of  Germany 
where  instruction  was  to  be  obtained  from  some  of  Koch's  earlier 
pupils.  But  to-day  bacteriological  laboratories  have  been  established 
in  all  parts  of  the  world,  and  it  is  no  longer  necessary  to  go  to  Ger- 
many to  obtain  such  instruction.  The  literature  of  the  subject  is. 
however,  largely  in  the  German  and  French  languages.  We  can 
only  refer  here  to  such  periodicals  as  are  principally  devoted  to  bac- 
teriological research  work. 

The  Zeitxchrift  fiir  Hygiene  has  been  published  since  1886,  and 
contains  numerous  valuable  papers,  contributed  for  the  most  part  bv 
the  pupils  of  Koch  and  of  Fliigge,  who  are  the  editors  of  the  journal. 

The  Annales  de  VInstitut  Pasteur  is  a  monthly  journal  which 
has  been  published  since  1888.  It  is  edited  by  Duclaux,  and  contains 
many  important  papers  and  reviews.  H-  well  as  the  statistics  of  the 


HISTORICAL.  9 

Pasteur  Institute  relating  to  preventive  inoculations  against  hydro- 
phobia. 

The  Annales  de  Micrographie  is  a  monthly  journal,  published  in 
Paris.  The  principal  editor  is  Miquel. 

The  Centralllalt  jur  Bakteriologie  und  Parasitenkunde  is  a 
weekly  journal  which  has  been  published  by  Gnstav  Fischer,  of  Jena, 
since  1887.  The  editors  are  Uhlworm,  of  Cassel;  Loffler,  at  present 
professor  a,t  Greifswald;  and  Leuckart,  professor  at  Leipzig. 

The  Journal  of  Pathology  and  Bacteriology  is  published 
monthly  in  Edinburgh  and  London.  It  dates  from  1892. 

A  most  important  work  for  students  of  bacteriology  is  the  Jalires- 
bertchtot  Baumgarten,  which  has  been  published  since  1885  by  Harald 
Bruhn,  Braunschweig,  Germany.  This  gives  a  brief  abstract  of 
nearly  ever}7  paper  of  importance  relating  to  the  subject  which  has 
been  published  during  the  year. 

The  Journal  of  Hygiene  is  a  new  quarterly,  edited  by  Dr.  George 
H.  F.  Nuttall,  and  published  in  Cambridge,  England.  In  the  first 
number  (January  1st,  1901)  the  accomplished  editor  says:  "The 
Journal  of  Hygiene  will  fulfil  a  definite  purpose  by  serving  as  a 
focus  to  English-speaking  investigators  for  work  in  Physics,  Chemis- 
try, Physiology,  Pathology,  Bacteriology,  Parasitology,  and  Epi- 
demiology, in  relation  to  Hygiene  and  Preventive  Medicine." 


II. 

CLASSIFICATION. 

THE  earlier  naturalists — Ehrenberg  (1838),  Dujardin  (1841) — 
placed  the  bacteria  among  the  infusoria;  but  they  are  now  recog- 
nized as  vegetable  microorganisms,  differing  essentially  from  the 
infusoria,  which  are  unicellular  animal  organisms.  One  of  the  prin- 
cipal points  in  differentiating  animal  from  vegetable  organisms 
among  the  lowest  orders  of  living  things  is  the  fact  that  animal 
organisms  receive  food  particles  into  the  interior  of  the  body,  assimi- 
lating the  nutritious  portion  and  subsequently  extruding  the  non- 
nutritious  residue  ;  vegetable  organisms,  on  the  other  hand,  are 
nourished  through  the  cell  wall  which  encloses  their  protoplasm,  by 
organic  or  inorganic  substances  held  in  solution. 

Ehrenberg  (1838),  under  the  name  of  vibrioiiiens,  established  four  gen- 
era, as  follows : 

1.  Bacterium — filaments  linear  and  inflexible. 

2.  Vibrio — filaments  linear,  snake-like,  flexible. 

3.  Spirillum — filaments  spiral,  inflexible. 

4.  Spirochcete — filaments  spiral,  flexible. 

Dujardin  (1841)  united  the  two  genera  Spirillum  and  Spirochcate  of 
Ehrenberg,  and  added  to  the  description  of  the  generic  characters  as  fol- 
lows: 

1.  Bacterium — filaments  rigid,  with  a  vacillating  movement. 

2.  Vibrio — filaments  flexible,  with  an  undulatory  movement. 

3.  Spirillum — filaments  spiral,  movement  rotatory. 

It  will  be  seen  that  this  classification  leaves  110  place  for  the  motionless 
bacilli,  such  as  the  anthrax  bacillus  and  many  others,  and  does  not  include 
the  spherical  bacteria,  now  called  micrococci. 

The  classification  of  Davaine  (18(38)  provides  for  the  motionless,  fila- 
mentous bacteria,  but  does  not  include  the  micrococci.  This  author  first 
insisted  that  the  vibrioniens  of  Ehrenberg  are  truly  vegetable  organisms, 
allied  to  the  algae.  He  makes  four  genera,  as  follows: 

Filaments    straight    or    bent,  L  Moving    spontane-  ]  Rigid  Bacterium. 
but  not  in  a  spiral,  ously,  }  Flexible  Vibrio. 

( Motionless,         .          Bacteridium. 
Filaments  spiral, Spirillum. 

Following  Davaine,  the  French  bacteriologists  frequently  speak  of  the 
motionless  anthrax  bacillus  as  la  bacteridie. 

Hoffman  in  1869  included  in  his  classification  the  spherical  bacteria, 
and  pointed  out  the  fact  that  motility  could  not  be  taken  as  a  generic  char- 
acter, as  it  was  not  constant  in  the  same  species  and  depended  to  some  ex- 
tent upon  temperature  conditions,  etc. 


CLASSIFICATION.  11 

Having  determined  that  the  bacteria  are  truly  vegetable  organ- 
isms, the  attention  of  botanists  has  been  given  to  the  question  as  to 
what  class  of  vegetable  organisms  they  are  most  nearly  related  to. 
There  are  decided  differences  of  opinion  in  this  regard.  While  Da- 
vaine,  Rabenhorst,  and  Cohn  insist  upon  their  affinities  with  the 
alga3,  Robin,  Nageli,  and  others  consider  them  fungi.  One  of  the 
principal  characters  which  distinguish  the  alga3  from  the  fungi  is 
the  presence  of  chlorophyll  in  the  former  and  its  absence  in  the  latter. 
Now,  the  bacteria  are  destitute  of  chlorophyll,  and  in  this  regard 
resemble  the  fungi;  yet  in  others  their  affinities  with  the  Palmellacece 
and  Oscillatoriacece  are  unmistakable.  It  is  not  necessary,  how- 
ever, that  we  should  consider  them  as  belonging  to  either  of  these 
classes  of  the  vegetable  kingdom.  By  considering  them  a  distinct 
class  of  unicellular  vegetable  organisms,  under  the  general  name  of 
bacteria,  we  may  avoid  the  difficulties  into  which  the  botanists  have 
fallen. 

We  must  refer  briefly,  however,  to  the  classification,  proposed  by  some 
of  the  leading-  German  botanists. 

Nageli,  placing1  the  bacteria  among1  the  lower  fungi,  which  give  rise  to 
the  decomposition  of  organic  substances,  divides  these  into  three  groups: 

1.  The  Mucorini,  or  mould  fungi. 

2.  The  Saccharomycetes,  or  budding  fungi,  which  produce  alcoholic  fer- 
mentation in  saccharine  liquids. 

3.  The  Schizomycetes,  or  fission  fungi,  which  produce  putrefactive  pro- 
cesses, etc. 

Cohn,  under  the  iiamo  of  Schizophytes,  has  grouped  these  low  vegetable 
organisms,  whether  provided  or  not  with  chlorophyll,  into  two  tribes  hav- 
ing the  following  characters: 

1.  GLJEOGENES — cells  free  or  united   into  glairy  families  by  an  intercel- 
lular substance. 

2.  NEMA.TOGEXES — cells  disposed  in  filaments. 

In  the  first  tribe  he  has  placed  the  genera  Micrococcus  (Hallier),  Bacte- 
rium (Dujardin),  Merismopedia  (Meyer),  Sarcina  (Goodsir),and  Ascococcus 
(Billroth),  with  various  genera  of  unicellular  algae  containing  chlorophyll. 

In  the  second  tribe  we  have  the  genera  Bacillus  (Cohn),  Leptothrix 
(Kg.),  Vibrio  (Ehr.),  Spirillum  (Ehr.),  Spirochcete  (Ehr.),  Streptococcus 
(Billr.),  Cladothrix  (Cohn),  and  Streptothrix  (Cohn),  associated  with  gen- 
era of  green  filamentous  algae. 

The  German  botanist  Sachs  unites  the  fungi  and  the  algae  into  a  single 
group,  the  Thallophytes,  in  which  he  establishes  two  parallel  series,  one  in- 
cluding those  containing  chlorophyll,  and  the  other  without,  as  follows: 


THALLOPHYTES. 

Forms  with  chlorophyll.  Forms  without  chlorophyll. 

Class  I. — Protophytes. 

A.  Cyanophyceae     (Oscillatoria-  A.  Schizomycetes  (Bacteria), 
ceae,  etc.). 

B.  Palmellaceae.  B.  Saccharomycetes. 


12  CLASSIFICATION. 

Zopf ,  Avho  insists  upon  the  polymorphism  of  these  low  organisms,  divides 
the  bacteria  into  four  groups: 

Genera. 
Streptococcus, 

1.  COCCOCE^:  — Up   to  the    pre-  Merismopedia, 
sent  time,  only  known  in  the  form  of  j  Sarcina, 
cocci.                                                                      Micrococcus, 

Ascococcus. 

2.  BACTERIACEJE. — Have  for  the  ]  Bacterium, 
most    part    spherical,    rod-like,    and   j  Spirillum, 
filamentous  forms  ;  the  first  (cocci)   (  Vibrio, 
may   be  wanting  ;   the  last  are  not  \  Leuconostoc, 
different  at  the  two  extremities;  fila-  Bacillus, 
ments  straight  or  spiral.  Clostridium. 

3.  LEPTOTRICHE.E.--  Spherical,  ]         Crmnthri'r 
rod-shaped,  and  filamentous  forms;  ^     l'Y™ 
the  last  show  a  difference  between  the 

two  extremities  ;   filaments  straight  |          Levtothrix 
orspiral ;  spore  formation  not  known.  J 

4.  CLADOTRICHE^.  --  Spherical,   | 
rod-shaped,  filamentous,  and    spiral   | 

forms  ;   the   filamentous    form    pre-   \  Cladothrix. 

sents  pseudo-branches  ;  spore  forma-   | 
tion  not  known. 

The  main  objection  to  this  classification  is  that  it  assumes  a  pleomorph- 
ism  for  the  bacteria  of  the  second  group — Bact^riaceoe — which  has  only  been 
established  for  a  few  species,  and  which  appear?  not  to  be  general  among  the 
rod  shaped  and  spiral  bacteria. 

De  Bary  divides  the  bacteria  into  two  principal  groups,  one  including 
those  which  form  endospores,  and  the  other  those  which  are  reproduced  by 
arthrospores.  But  our  knowledge  is  yet  too  imperfect  to  make  this  classifi- 
cation of  value,  and  the  same  may  be  said  of  Hueppe's  recent  attempt  at 
classification,  in  which  the  mode  of  reproduction  is  a  principal  feature. 

The  classification  of  Baumgarten  (1890)  appears  to  us  to  have 
more  practical  value,  and,  with  slight  modifications,  we  shall  adopt 
it  in  the  present  volume.  This  author  divides  the  bacteria  into  two 
principal  groups,  as  follows  : 

GROUP  I.  Species  relatively  monomorphous. 

GROUP  II.  Species  pleomorphous. 

The  first  group  includes  the  micrococci,  the  bacilli,  and  the 
spirilla;  the  second  group  the  spirulina  of  Hueppe,  leptotrichece 
(Zopf),  and  cladotrichece. 

The  pleomorphous  species  described  by  Hauser  under  the  generic 
name  Proteus  are  included  in  the  second  group  among  the  spirulina. 
In  the  present  volume  we  have  described  these  pleomorphous  species 
among  the  bacilli. 

The  Cocci,  in  the  classification  of  Baumgarten,  constitute  a  single 
genus  with  the  following  subgenera  :  1,  Diplococcus  j  2,  Strepto- 
coccus ;  3,  Merismopedia  (Zopf) — "Merista"  (Hueppe);  4,  Sar- 
cina (Goodsir)  ;  5,  Micrococcus  ("  staphylococci"). 

The  BACILLI  are  included  in  a  single  genus  embracing  all  of 


CLASSIFICATION.  13 

those  species  which  only  form  rod-shaped  cells,  and  filaments  com- 
posed of  rod-like  segments  ;  or  straight  filaments  not  distinctly  seg- 
mented, which  may  be  rigid  or  flexible. 

The  SPIRILLA  are  also  included  in  a  single  genus,  embracing  all 
of  those  species  in  which  the  filaments  are  spiral  in  form  and  the 
segments  more  or  less  spiral  or  ''comma-shaped  "  —filaments  either 
rigid  or  flexible. 

This  simple  morphological  classification  of  the  monomorphous 
group  of  Baumgarten  corresponds  with  the  nomenclature  now  gene- 
rally in  use  among  bacteriologists.  We  speak  of  the  spherical  bac- 
teria as  cocci  or  as  micrococci,  of  the  rod-shaped  bacteria  as  bacilli, 
and  of  the  spiral  bacteria  as  spirilla. 

It  is  true,  however,  that  we  are  sometimes  embarrassed  to  decide 
whether  a  particular  microorganism  belongs  to  one  or  the  other  of 
these  morphological  groups  or  so-called  genera.  Among  the  bacilli, 
for  example,  we  may  have,  in  the  same  pure  culture,  rods  of  very 
different  lengths,  some  being  so  short  that  if  alone  they  might  be 
taken  for  cocci,  while  others  may  have  grown  out  into  long  fila- 
ments. But  if  we  are  assured  that  the  culture  is  pure  the  presence 
of  rod  forms  establishes  the  diagnosis,  and  usually  the  cocci-like 
elements,  when  carefully  observed,  will  be  seen  to  be  somewhat 
longer  in  one  diameter  than  in  the  other.  The  German  bacterio- 
logists generally  insist  upon  placing  among  the  bacilli  all  straight  bac- 
teria in  which,  as  a  rule,  one  diameter  is  perceptibly  greater  than 
that  transverse  to  it ;  and  several  species  of  well-known  bacteria 
which  were  formerly  classed  as  micrococci  a-re  now  called  bacilli — 
e.g.,  Friedlander's bacillus  ("  pneumococcus"),  Bacillus prodigio&us. 

The  distinction  made  by  Colin  and  others  between  the  genus 
Bacterium  (Duj.)  and  the  genus  Bacillus  (Colin)  cannot  be  main- 
tained, inasmuch  as  we  may  have  short  rods  and  quite  long  fila- 
ments in  the  same  pure  culture  of  a  single  species  ;  and,  again,  the 
character  upon  which  the  genus  Vibrio  (Ehr.)  was  established — 
viz.,  the  fact  that  the  filaments  are  flexible  and  the  movements 
sinuous — is  not  a  sufficient  generic  or  even  specific  character,  for  in 
a  pure  culture  there  may  be  short  rods  which  are  rigid,  and  long 
filaments  which  are  flexible  and  hjave  a  sinuous  movement.  We 
therefore  to-day  speak  of  all  the  elongated  forms  as  bacilli,  unless 
they  are  spiral  and  have  a  corkscrew-like  motion,  in  which  case  they 
are  known  as  spirilla. 

f  The  bacteria  are  also  classified  according  to  their  biological  char- 
acters, and  it  will  be  necessary  to  consider  the  various  modes  of 
grouping  them  from  different  points  of  view  other  than  that  which 
relates  to  their  form.  This  is  the  more  important  inasmuch  as  we 
are  not  able  to  differentiate  species  by  morphological  characters 


14  CLASSIFICATION. 

alone.  Thus,  for  example,  there  are  among  the  spherical  bacteria,  or 
micrococci,  numerous  well-established  species  which  the  most  expert 
microscopist  could  not  differentiate  by  the  use  of  the  microscope 
alone  ;  the  same  is  true  of  the  rod-shaped  bacteria.  The  assump- 
tion often  made  by  investigators  who  are  riot  sufficiently  impressed 
with  this  fact,  that  two  microorganisms  from  different  sources,  or 
even  from  the  same  source,  are  the  same  because  stained  prepara- 
tions examined  under  the  microscope  look  alike,  has  led  to  serious 
errors  and  to  much  confusion.  As  an  example  of  what  is  meant  we 
may  refer  to  the  pus  organisms.  Before  the  introduction  of  Koch's 
"plate  method"  micrococci  had  been  observed  in  the  pus  of  acute 
abscesses.  Some  of  these  were  grouped  in  chains — streptococci— 
and  some  were  single,  or  in  pairs,  or  in  groups  of  four ;  but  whether 
these  were  simply  different  modes  of  grouping  in  a  single  species,  or 
whether  the  chain  micrococci  represented  a  distinct  species,  was  not 
determined  with  certainty.  That  there  were  in  fact  four  or  more 
distinct  species  to  be  found  in  the  pus  of  acute  abscesses  was  not 
suspected  until  Rosenbach  and  Passet  demonstrated  that  this  is  the 
case,  and  showed  that  not  only  is  the  streptococcus  a  distinct  species, 
but  that  among  the  cocci  not  associated  in  chains  there  are  three 
species  which  are  to  be  distinguished  from  each  other  by  their  color 
when  grown  on  the  surface  of  a  solid  culture  medium.  One  of  these 
has  a  milk-white  color,  one  is  of  a  lemon-yellow  color,  while  the  third 
is  a  golden-yellow. 

Those  microorganisms  which  form  pigment  are  called  chromo- 
genes,  or  chromogenic ;  those  which  produce  fermentations  are 
spoken  of  as  zymogenes,  or  zymogenic  ;  those  which  give  rise  to  dis- 
ease processes  in  man  or  the  lower  animals  are  denominated  patho- 
genes,  or  pathogenic.  We  cannot,  however,  classify  bacteria  under 
the  three  headings  chromogenes,  zymogenes,  and  pathogenes,  for 
some  of  the  chromogenic  species  are  also  pathogenic,  as  are  some 
of  the  zymogenes.  These  characters  must  therefore  be  considered 
separately  as  regards  each  species,  and  in  studying  its  life  history  and 
distinguishing  characters  we  determine  whether  it  is  chromogenic 
or  non-chromogenic ;  whether  it  produces  special  fermentations ; 
and  whether  it  is  or  is  not  pathogenic  when  inoculated  into  the 
lower  animals.  In  making  the  distinction  between  pathogenic 
and  non-pathogenic  microorganisms  we  must  remember  that  a 
certain  species  may  be  pathogenic  for  one  animal  and  not  for  an- 
other. Thus  the  anthrax  bacillus,  which  is  fatal  to  cattle,  sheep, 
rabbits,  guinea-pigs,  and  mice,  does  not  kill  white  rats  ;  the  bacillus 
of  mouse  septicaemia  kills  house  mice,  but  field  mice  are  fully  im- 
mune from  its  pathogenic  effects  ;  on  the  other  hand,  the  bacillus  of 
glanders  is  fatal  to  field  mice  but  not  to  house  mice. 


CLASSIFICATION.  15 

Again,  it  must  be  remembered  that  pathogenic  power  also  de- 
pends, to  a  greater  or  less  extent,  upon  the  dose  injected  into  an 
animal  as  compared  to  its  body  weight.  Some  pathogenic  organ- 
isms in  very  minute  doses  give  rise  to  a  fatal  infectious  malady ; 
others  are  only  able  to  overcome  the  vital  resisting  power  of  the 
tissues  and  fluids  of  the  body  when  introduced  into  the  circulation, 
or  into  the  subcutaneous  tissue  or  abdominal  cavity,  in  considerable 
amounts.  Some  pathogenic  bacteria  invade  the  blood  ;  others  mul- 
tiply only  in  certain  tissues  of  the  body  ;  and  others  again  multiply 
in  the  intestine  and  by  the  formation  of  poisonous  products  which 
are  absorbed  show  their  pathogenic  power. 

Another  classification  of  the  bacteria  relates  to  the  environment 
favorable  to  their  development.  Thus  we  speak  of  saprophytic  and 
parasitic  bacteria,  or  of  SAPROPHYTES  and  PARASITES. 

The  saprophytes  are  such  as  exist  independently  of  a  living  host, 
obtaining  their  supply  of  nutriment  from  dead  animal  or  vegetable 
material  and  from  water  containing  organic  and  inorganic  matters 
in  solution.  The  strict  parasites,  on  the  other  hand,  depend  upon 
a  living  host,  in  the  body  of  which  they  multiply,  sometimes  without 
injury  to  the  animal  upon  which  they  depend  for  their  existence,  but 
frequently  as  harmful  invaders  giving  rise  to  acute  or  chronic  infec- 
tious diseases.  Microorganisms  which  ordinarily  lead  a  saprophy- 
tic existence,  but  which  can  also  thrive  within  the  body  of  a  living 
animal,  are  called  facultative  parasites.  Thus  the  leprosy  bacillus, 
which  is  only  found  in  leprous  tissues,  is  a  strict  parasite  ;  while  the 
typhoid  bacillus,  the  cholera  spirillum,  etc. ,  are  facultative  parasites, 
inasmuch  as  they  are  capable  of  maintaining  an  independent  exist- 
ence, for  a  time  at  least,  external  to  the  bodies  of  living  animals. 

It  seems  probable  that  the  pathogenic  organisms  which  are  only 
known  to  us  to-day  as  strict  parasites  were,  at  some  time  in  the  past, 
saprophytes,  which  gradually  became  accustomed  to  a  parasitic 
mode  of  existence,  and,  under  the  changed  conditions  of  their  envi- 
ronment, finally  lost  the  power  of  living  in  association  with  other 
saprophytes  exposed  to  variations  of  temperature,  etc.  The  tubercle 
bacillus,  for  example,  is  known  to  us  only  as  a  parasite  which  has  its 
habitat  in  the  lungs,  lymphatic  glands,  etc. ,  of  man  and  of  certain 
of  the  lower  animals.  But  we  are  able  to  cultivate  it  in  artificial 
media  external  to  the  body  ;  and  it  is  in  accord  with  modern  views 
relating  to  the  development  of  species  to  suppose  that  at  some  time 
in  the  past  it  was  able  to  lead  a  saprophytic  existence.  Not  to  admit 
this  forces  us  to  the  conclusion  that,  at  some  time  subsequent  to  the 
appearance  of  man  and  the  lower  animals  in  which  it  is  now  found 
as  a  parasite,  it  was  created  with  its  present  biological  characters, 
which  restrict  it  to  a  parasitic  existence  in  the  bodies  of  these  ani- 


16  CLASSIFICATION. 

mals,  and  that,  consequently,  the  immense  destruction  of  human  life 
which  has  resulted  from  its  parasitic  invasion  of  successive  genera- 
tions was  designed  when  it  was  created.  The  opposite  view  is  sup- 
ported by  numerous  facts  which  show  that  these  low  organisms,  like 
those  higher  in  the  scale,  are  subject  to  modifications  as  a  result  of 
changed  conditions  of  environment,  and  that  such  modifications,  in 
the  course  of  time,  may  become  well-established  specific  characters. 

Again,  the  bacteria  may  be  grouped  into  aerobic  and  anaerobic 
species.  This  is  a  very  important  distinction,  which  was  first  estab- 
lished by  Pasteur,  who  found  that  certain  bacteria  will  only  grow 
when  freely  supplied  with  oxygen,  while  others  absolutely  decline  to 
grow  in  the  presence  of  this  gas.  The  latter,  which  are  spoken  of  as 
strict  anaerobics,  may  be  cultivated  in  a  vacuum  or  in  an  atmo- 
sphere of  hydrogen.  Those  species  which  grow  either  in  the  pre- 
sence of  oxygen  or  when  it  is  excluded  are  called  facultative  an- 
aerobics. 

Certain  bacteria  produce  a  peptonizing  ferment  which  has  the 
power  of  liquefying  gelatin.  This  has  led  to  the  classification  of 
those  microorganisms  of  this  class  which  grow  in  Koch's  flesh-pep- 
tone-gelatin as  liquefying  and  non-liquefying  bacteria. 

Again,  we  speak  of  them  as  motile  or  non-motile. 

It  is  evident  that  these  biological  characters,  although  all-im- 
portant in  the  definition  of  species,  cannot  serve  us  in  an  attempt  to 
establish  natural  genera  ;  for  the  lines  a?e  not  sharply  drawn  between 
the  saprophytes  and  the  parasites,  the  aerobics  and  the  anaerobics, 
etc. ,  inasmuch  as  we  have  facultative  parasites  and  facultative  an- 
aerobics which  we  cannot  include  in  either  class,  and  which  yet  do 
not  form  a  distinct  class  by  themselves.  We  therefore  adhere  to  the 
morphological  classification,  although  this  is  open  to  criticism.  For 
example,  among  the  rod-shaped  organisms  which  we  call  bacilli  and 
describe  under  the  generic  name  Bacillus  there  are  some  which 
multiply  by  binary  division  only,  while  others  form  endogenous  re- 
productive bodies  known  as  spores.  Certainly  so  important  a  differ- 
ence in  the  mode  of  reproduction  should  be  sufficient  to  separate 
these  rod-shaped  organisms  into  two  natural  groups  or  genera. 

As  heretofore  stated,  the  German  bacteriologist  Hueppe  has  at- 
tempted a  classification  based  upon  the  mode  of  reproduction,  in 
which  he  makes  two  groups,  or  "tribes/'  one  in  which  reproduction 
occurs  by  the  formation  of  endogenous  spores — •"  endospores  "  —the 
other  in  which  it  occurs  by  the  formation  of  "  arthrospores."  The 
latter  group  includes  all  of  those  bacteria  in  which  no  other  mode  of 
multiplication  is  known  than  that  by  binary  division,  which  is  com- 
mon to  all.  In  the  present  state  of  our  knowledge  this  classification 
•  '  An  account  of  this  mode  of  reproduction  is  given  on  page  19. 


CLASSIFICATION.  17 

is  scarcely  to  be  considered  of  practical  value,  inasmuch  as  the  ques- 
tion of  spore  formation  is  still  undetermined  for  a  large  number  of 
species. 

In  the  following  table  we  shall  give  the  characters  of  the  dif- 
ferent genera  which  have  been  described  by  recent  botanists  and 
bacteriologists,  arranged  under  the  three  headings,  MiCROCOCCi, 
BACILLI,  SPIRILLA.  Where  we  doubt  the  propriety  of  maintaining 
a  distinct  generic  name  upon  the  supposed  distinguishing  characters, 
the  description  will  be  printed  in  small  type. 

MICROCOCCI. 

General  Characters. — Spherical  bacteria  which  are  reproduced 
by  binary  division  ;  usually  without  spontaneous  movements  ;  do  not 
form  endogenous  spores.  (According  to  some  authors,  certain  cells, 
known  as  arthrospores,  may  be  distinguished  by  their  greater  size 
and  refractive  power,  and  these  are  supposed  to  have  greater  resist- 
ance to  desiccation  than  the  ordinary  cocci  resulting  from  binary 
division,  and  to  serve  as  reproductive  bodies. )  Some  micrococci  are 
not  precisely  round,  but  are  somewhat  oval  in  form  ;  and  when  in 
process  of  division  the  cocci,  necessarily,  are  more  or  less  elongated 
in  one  diameter  before  a  complete  separation  into  two  spherical  ele- 
ments has  occurred. 

MiCROCOCCUS. — Division  in  one  direction ;  cocci  single,  in  pairs, 
or  accidentally  associated  in  irregular  groups  ;  sometimes  held  to- 
gether in  irregular  masses  by  a  transparent,  glutinous,  intercellular 
substance.  (Micrococci  belonging  to  this  genus  are  frequently  de- 
scribed as  "  staphylococci,"  and  Staphylococcus  is  used  by  Rosen- 
bach  as  a  generic  name  for  the  pus  cocci  described  by  him,  which 
are  solitary  or  associated  in  irregular  groups,  as  above  described. ) 

Ascococcus. — Cocci  associated  in  globular  or  lobulated,  zooglcea 
masses  by  a  rather  firm  intercellular  substance. 

LEUCOXOSTOC. — Cocci,  solitary  or  in  chains,  surrounded  by  a 
thick,  gelatinous  envelope  and  forming  zoogloea  of  cartilaginous 
consistence. 

STREPTOCOCCUS. — Division  in  one  direction  only ;  cocci  associ- 
ated in  chains. 

Diplococcus. — Division  in  one  direction  only  ;  cocci  associated  in  pairs. 

Association  in  pairs  is  common  to  all  of  the  micrococci,  inasmuch  as 
they  multiply  by  binary  division.  When  such  association  has  rather  a  per- 
manent character,  it  is  customary  to  speak  of  the  microorganism  as  a  diplo- 
coccus,  but  we  doubt  the  propriety  of  recognizing-  this  mode  of  association 
as  a  generic  character. 

MERISMOPEDIA. — Division  in  two  directions,  forming  groups  of 
four,  which  remain  associated  in  a  single  plane — "tetrads." 

SARCINA. — Division  in  three  directions,  forming  packets  of  eight 
2 


18  CLASSIFICATION. 

or  more  elements,  which  remain  associated  in  more  or  less  regular 
cubical  masses. 

BACILLI. 

General  Characters. — Rod-shaped  and  filamentous  (not  spiral) 
bacteria  in  which  there  is  no  differentiation  between  the  extremities 
of  the  rods  ;  reproduction  by  binary  division  in  a  direction  trans- 
verse to  the  long  axis  of  the  rods,  or  by  binary  division  and  the  for- 
mation of  endogenous  spores  ;  rigid  or  flexible  ;  motile  or  non-motile. 

BACILLUS. — Characters  as  given  above. 

Bacterium. — This  genus,  established  by  Dujardin,  is  now  generally 
abandoned,  the  species  formerly  included  in  it  being  transferred  to  the  genus 
Bacillus.  As  denned  by  Cohn,  the  generic  characters  were  :  Cells  cylindri- 
cal or  elliptical,  free  or  united  in  pairs  during  their  division,  rarely  in 
fours,  never  in  chains,  sometimes  in  zoogloea  (differing  from  the  zooglcea 
of  spherical  bacteria  by  a  more  abundant  and  firmer  intercelluar  substance), 
having  spontaneous  movements,  oscillatory  and  very  active,  especially  in 
media  rich  in  alimentary  material  and  in  presence  of  oxygen. 

Clostridium. — Rod-shaped  bacteria  which  form  large,  endogenous,  and 
usually  oval  spores  ;  these  are  centrally  located,  and  during  the  stage  of 
spore  formation  the  rods  become  fusiform. 

SPIRILLA. 

General  Characters. — Curved  rods  or  spiral  filaments ;  rigid  or 
flexible  ;  reproduction  by  binary  division,  or  by  binary  division  and 
the  formation  of  endogenous  spores  (or  by  arthrospores  ?) ;  move- 
ments rotatory  in  the  direction  of  the  long  axis  of  the  filaments. 

SPIRILLUM. — Characters  as  above. 

Spirochcete. — Flexible,  spiral  filaments;  movements  rotatory. 

Vibrio, — Filaments  flexible,  straight  or  sinuous;  movements  sinuous. 

A  considerable  number  of  bacteria  which  are  usually  seen  as  short,  curved 
rods,  but  which  may  grow  out  into  long,  spiral  filaments,  are  described  by 
some  authors  under  the  generic  name  Vibrio,  e.g.,  the  so-called  "comma 
bacillus'1  of  Koch — "  Spirillum  cholerae  Asiaticae";  the  spirillum  of  Finkler 
and  Prior — "  Vibrio  proteus" ;  the  spirillum  described  by  Gameleia — "Vibrio 
Metschnikovi,"etc.  These  microorganisms  have  not  the  characters  which 
distinguished  the  genus  Vibrio  as  established  by  Ehrenberg,  and  we  prefer  to 
follow  Fliigge  in  describing  them  under  the  generic  name  Spirillum. 

The  pathogenic  bacteria  now  known  belong  to  one  or  the  other 
of  the  above-described  genera,  and  the  attention  of  bacteriologists 
has  been  given  chiefly  to  the  study  of  micrococci,  bacilli,  and  spirilla. 
But  the  botanists  place  among  the  bacteria  certain  other  forms  which 
are  found  in  water,  and  which,  in  a  systematic  account  of  this  class 
of  microorganisms,  demand  brief  attention  at  least.  These  are  in- 
cluded in  Baumgarten's  second  group,  which  includes  the  pleomor- 
phous  bacteria. 

SPIRULINA  (Hueppe). — The  vegetative  cells  are  sometimes  rod- 
shaped  and  sometimes  spiral ;  in  suitable  media  they  may  grow  out 


CLASSIFICATION.  19 

into  long,  straight,  wavy,  or  spiral  filaments.     These  filaments  may 
break  up  into  cocci-like  reproductive  elements — 4<  arthrospores." 

LEPTOTRICHE.E  (Zopf). — The  vegetative  cells  present  rod-shaped 
and  spiral  forms,  and  grow  out  into  straight,  wavy,  or  spiral  fila- 
ments ;  these  may  show  a  difference  between  the  two  extremities, 
of  base  and  apex.  Cocci-like  reproductive  bodies  are  formed  by  seg- 
mentation of  the  rod-shaped  elements  in  these  filaments.  In  some 
of  the  species  the  segments  are  enclosed  in  a  common  sheath.  Sub- 
genera:  LEPTOTHRIX,  BEGGIATOA,  CREXOTHRIX,  PHRAGMIDIO- 
THRIX  (for  generic  characters  see  page  12). 

CLADOTRICHE^E  (Zopf). — The  vegetative  cells  are  rod-shaped 
or  spiral,  and  grow  out  into  straight  or  spiral  filaments,  which  may 
present  pseudo-ramifications.  A  single  genus,  CLADOTHRIX  (see 
page  12). 

The  various  methods  of  classification  heretofore  referred  to  must 
all  be  considered  provisional  and  unsatisfactory  from  a  scientific 
point  of  view.  Thus  Hueppe  says :  "  The  existence  of  rigid  form 
species,  which  not  only  the  earlier  observers,  but  even  Cohn,  §chroter, 
and  Koch  assumed,  can  be  upheld  no  longer.  The  adaptability  of 
bacterial  forms  to  changing  conditions  of  nutrition  is  not  so  bound- 
less as  Naegeli  and  Billroth  supposed,  but  it  is  considerably  greater 
than  was  once  held  to  be  compatible  with  the  conception  of  the  ex- 
istence of  constant  species." 

A.  Fischer  has  attempted  to  make  use  of  the  presence,  number, 
and  mode  of  attachment  of  flagella  as  a  means  of  classification.  No 
doubt  this  character  and  the  presence  or  absence  of  spores  should  re- 
ceive consideration  in  any  attempt  at  a  scientific  classification  of  the 
bacteria. 


III. 

MORPHOLOGY. 

IN  the  present  chapter  we  shall  give  a  general  account  of  the 
morphology,,  modes  of  grouping,  and  dimensions  of  the  bacteria. 

The  standard  of  measurement  used  by  bacteriologists  is  the  micro- 
millimetre,  or  the  one-thousandth  part  of  a  millimetre.  This  is 
represented  by  the  Greek  letter  //.  One  ^  (micromillimetre)  is  equal 
to  about  one-twenty-five-thousandth  of  an  English  inch. 

The  spherical  bacteria,  or  micrococci,  differ  greatly  in  size,  and 
also  in  the  mode  of  grouping  when,  as  a  result  of  binary  division, 
they  remain  associated  one  with  another.  The  smallest  may  mea- 
sure no  more  than  0. I//,  while  some  of  the  larger  species  are  from 
one  to  two  ^  in  diameter.  The  enormous  number  of  these  minute 
organisms  which  may  be  contained  in  a  small  drop  of  a  pure  culture 
may  be  easily  estimated  in  a  rough  way.  Compare  a  single  micro- 
coccus,  for  example,  with  a  sphere  having  a  diameter  of  one-twenty- 
fifth  of  an  inch.  If  our  micrococcus  is  one  of  the  larger  sort,  having 
a  diameter  of  one  //,  it  would  take  a  chain  of  one  thousand  to  reach 
across  the  diameter  of  such  a  sphere,  and  its  mass,  as  compared 
to  the  larger  sphere,  would  be  as  1  to  523,600,000. 

The  number  of  cocci  in  a  milligramme  of  a  pure  culture  of  Staphy- 
lococcus  pyogenes  aureus  has  been  estimated  by  Bujwid,  by  count- 
ing, at  8,000,000,000. 

Not  only  do  different  species  differ  in  dimensions,  but  consider- 
able differences  in  size  may  be  recognized  in  the  individual  cocci  in  a 
pure  culture  of  the  same  species.  On  the  other  hand,  there  are 
numerous  species  which  so  closely  resemble  each  other  in  size  and 
mode  of  association  that  they  cannot  be  differentiated  b}^  a  micro- 
scopic examination  alone,  and  we  must  depend  upon  other  characters, 
such  as  color,  growth  in  various  culture  media,  pathogenic  power, 
etc. ,  to  decide  the  question  of  identity  or  non-identity. 

When  in  active  growth  the  micrococci  necessarily  depart  from  a 
typical  spherical  form  just  before  dividing,  and  under  these  circum- 
stances may  be  of  a  short  or  long  oval.  When  division  has  taken 
place,  if  the  two  members  of  a  pair  remain  associated  they  are  often 
more  or  less  flattened  at  the  point  of  contact  (Fig.  1,  a). 


MORPHOLOGY. 


When  in  a  culture  the  cocci  are  for  the  most  part  associated  in 
pairs  (Fig.  1,  d),  we  speak  of  the  organism  as  a  diplococcus. 

The  staphylococci  are  characterized  by  the  fact  that,  for  the  most 
part,  the  individual  cocci  in  a  culture  are  solitary  (Fig.  1,  6).  But, 
inasmuch  as  multiplication  occurs  by  binary  division,  we  also  have 
pairs  and  occasionally  a  group  of  four — probably  from  the  accidental 
apposition  of  two  pairs  (Fig.  1,  c) ;  or  they  may  be  associated  in  grape- 


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b 


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FIG.  1. 


like  bunches  ;  and  after  staining  and  mounting  a  preparation  we  find 
the  cells  associated  in  irregular  groups.  This  results  from  the  fact 
that  they  are  surrounded  by  a  glutinous  material  which  causes  them 
to  adhere  to  each  other  (Fig.  1,  e).  A  mass  of  cocci  held  together  in 


FIG.  2. 


FIG.  3. 


FIG.  4. 


this  way  by  a  transparent,  glutinous,  intercellular  substance  is  spoken 
of  as  a  zoogloea  (Fig.  2).  In  the  genus  Ascococcus  the  intercellar 
substance  is  quite  firm  and  the  zooglcea  are  in  the  form  of  spherical 
or  irregularly  lobulated  masses  surrounded  by  a  resistant  envelope  of 
jelly-like  material  (Fig,  3). 

When,  as  a  result  of  division  in  one  direction   only,  the  cocci 


22  MORPHOLOGY. 

remain  united  in  chains  (Fig.  4,  a),  they  are  described  as  streptococci, 
and  are  sometimes  spoken  of  as  in  chaplets  or  in  torula  chains.  In 
such  chains  we  frequently  find  the  evidence  of  recent  division  of  the 
cocci,  as  shown  by  the  grouping  of  the  elements  of  the  chain  into 
pairs  (Fig.  4,  b). 

When  division  occurs  habitually  in  two  directions,  groups  of  four 
result,  which  are  spoken  of  as  tetrads.  This  is  the  distinguishing 
character  of  the  genus  Merismopedia.  In  these  groups  of  four  the 
individual  cocci  are  often  flattened  at  the  points  of  contact,  as  in 
Fig.  5,  b.  We  also  find  pairs  and  groups  of  three  in  pure  cultures  of 
species  belonging  to  this  genus,  as  shown  in  Fig.  5,  c.  In  these, 
transverse  division  has  not  yet  occurred  in  one  or  in  both  elements  of 
a  pair.  This  association  of  micrococci  in  tetrads  seems  to  be  main- 
tained, in  some  species  at  least,  by  the  fact  that  each  group  of  four  is 
enclosed  in  a  jelly-like  capsule.  The  extent  of  this  capsule  differs  in 
the  same  species  under  different  circumstances;  as  a  rule,  it  is  most 
apparent  when  a  culture  has  been  made  in  a  liquid  medium.  Some  of 


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e 

FIG.  5. 


FIG 


the  diplococci  have  a  similar  capsule.  The  jelly-like  substance  does 
not  stain  well  with  the  aniline  colors  and  is  seen  as  a  transparent 
halo  around  the  stained  cocci.  Some  authors  (Frankel  and  Pfeiffer) 
believe  that  this  capsule  is  formed  by  the  swelling  up  of  the  cell 
membrane  as  a  result  of  the  imbibition  of  water. 

When  division  occurs  in  three  directions  packets  of  eight  or 
more  elements  are  formed.  This  mode  of  association  characterizes 
the  genus  Sarcina.  The  "packet  form "  is  best  seen  in  an  un- 
stained preparation  from  a  fresh  culture,  in  which  a  little  material 
suspended  in  water  is  examined  under  a  comparatively  low-power 
objective — one-sixth  (Fig.  6). 

Among  the  bacilli  there  is  room  for  a  wider  range  of  morphologi- 
cal characters.  They  differ  not  only  in  dimensions  and  in  modes  of 
grouping,  but  in  form.  The  relation  of  the  transverse  to  the  longi- 
tudinal diameters  affords  a  great  variety  of  forms,  varying  from  a 
short  oval  element  to  a  slender  rod  or  elongated  filament.  But  it 
must  be  remembered  that  we  may  have  short  rods  and  long  filaments 
in  a  pure  culture  of  the  same  bacillus— the  typhoid  bacillus,  for 


MORPHOLOGY. 


23 


example.  There  are  also  considerable  differences  in  the  transverse 
diameter  of  bacilli  belonging  to  the  same  species  when  cultivated  in 
different  media,  or  even  .  in  the  same  medium,  although,  as  a  rule, 
the  transverse  diameter  is  tolerably  uniform  in  pure  cultures. 

Again,  the  form  of  the  extremities  of  the  rods  is  to  be  observed 
(Fig.  7).  This  may  be  square,  or  the  corners  may  be  slightly 
rounded,  or  the  extremities  may  be  quite  round  or  lance-oval,  or 
the  outlines  of  the  rod  may  be  spindle-shaped  from  the  formation  of 


OO 


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Fio. 


a  large  central  spore — "cloatridium" — or  one  end  may  be  dilated 
from  the  formation  of  a  large  terminal  spore. 

In  old  cultures  we  frequently  find  irregular  forms  due  to  swellings 
and  constrictions,  which  probably  occur  in  bacilli  which  have  but 
little  vitality  or  are  already  dead.  These  are  spoken  of  as  involution 
forms  (Fig.  8). 

The  bacilli  multiply  by  binary  division  in  a  direction  transverse 
to  the  longitudinal  axis,  and,  as  a  result  of  such  binary  division,  long 


FIG.  9. 

chains  in  which  the  elements  remain  associated  may  be  formed 
(Fig.  9)  ;  or  the  rods  may  be  for  the  most  part  solitary  or  united  in 
pairs.  Like  the  micrococci,  the  bacilli  are  sometimes  surrounded  by 
a  gelatinous  envelope  or  capsule.  They  may  also  be  united  by  a 
glutinous  material  into  zoogloea  masses. 

Bacilli  which  under  certain  conditions  are  seen  as  short  rods 
may,  under  other  circumstances,  grow  out  into  long  filaments,  and 
these  may  be  associated  in  bundles  or  in  tangled  masses. 

The  spirilla  differ  from  the  bacilli  in  the  form  of  the  rods  and  fila- 


MORPHOLOGY. 


ments,  which  are  curved  or  spiral.  The  shorter  elements  in  a  pure 
culture  may  be  simply  curved,  as  in  a,  Fig.  10,  while  the  spiral  form 
becomes  apparent  in  those  which  are  longer,  and  we  may  have  one 
or  several  turns  of  the  spiral  (Fig.  10,  b).  The  spiral  form  may  be 
but  slightly  marked  (Fig.  10,  c),  or  the  turns  may  be  close  and  deep 
as  in  a  corkscrew  (Fig.  10,  d).  Again,  the  curved  filaments  may  be 
short  and  rigid,  or  long  and  flexible  (Fig.  10,  e). 

In  the  genus  Cladothrix,  which  is  placed  by  botanists  among 
the  bacteria,  the  filaments  appear  to  branch ;  but  this  branching  is 
only  apparent,  and  there  is  no  true  dichotomous  branching  in  this 
class  of  microorganisms.  The  false  branching  of  Cladothrix 
dichotoma,  Cohn,  is  shown  in  Fig.  11.  The  fact  that  some  of  the 
larger  species  of  bacilli  and  spirilla  are  provided  with  slender,  whip- 
like  appendages  called  flagella  has  been  known  for  many  years,  and 
it  has  for  some  time  been  suspected  that  all  of  the  motile  organisms 


FIG.  10. 


FIG.  11. 


FIG.  12. 


of  this  class  are  provided  with  similar  appendages  and  that  these  are 
organs  of  locomotion.  Recently,  by  improvements  in  methods  of 
staining,  Loffler  has  demonstrated  the  presence  of  flagella  in  many 
species  in  which  they  had  heretofore  escaped  observation.  They  are 
sometimes  single,  at  the  ends  of  the  rods  (Fig.  12,  a);  or  there  may 
be  several  at  the  extremity  of  a  single  rod  (Fig.  12,  6);  again,  they 
are  seen  in  considerable  numbers  around  the  periphery  of  the  rod 
(Fig.  12,  c). 

The  bacilli  and  spirilla  sometimes  contain  in  the  interior  of  the 
cells  granules  of  different  kinds.  These  may  appear  like  little  oil 
drops  or  they  may  be  more  opaque.  In  the  genus  Beggiatoa  grains 
of  sulphur  are  found  in  the  interior  of  the  cells.  Again,  we  may 
find  vacuoles  in  the  protoplasm  ;  or,  in  stained  preparations,  deeply 
stained  granules,  which  are  not  spores,  may  be  seen  at  the  extremi- 
ties of  the  rods — end-staining.  The  morphological  characters  de- 
pending upon  the  formation  of  endogenous  spores  will  be  referred  to 
hereafter. 


IV. 

STAINING  METHODS. 

THE  rapid  development  of  our  knowledge  with  reference  to  the 
minute  microorganisms  under  consideration  depends  very  largely 
upon  the  discovery  that  they  may  be  stained  by  various  dyes,  and  es- 
pecially by  the  aniline  colors.  Weigert  (1876)  was  the  first  to  employ 
these  colors  in  studying  the  bacteria,  and  Koch  at  once  recognized 
the  value  of  the  method  and  made  use  of  it  in  his  researches. 

The  basic  aniline  colors  are  those  employed,  and  among  these  the 
most  useful  are  fuchsin,  methylene  blue,  gentian  violet,  Bismarck 
brown,  and  vesuvin. 

Staining  upon  the  Cover  Glass  or  Slide. — By  a  "cover-glass 
preparation "  we  mean  that  material  supposed  to  contain  bacteria 
has  been  spread  out  upon  a  thin  glass  cover,  dried,  and  stained  for 
microscopical  examination.  A  small  drop  of  a  liquid  culture  may,  for 


FIG.  13. 


example,  be  spread  upon  a  perfectly  clean  cover  glass  by  means  of  a 
platinum  wire  held  in  a  glass  handle  (Fig.  13).  Or  we  may  place  a 
drop  of  water  in  the  centre  of  the  thin  glass  cover,  and  by  means  of 
the  same  instrument  take  a  little  material  from  a  culture  made  upon 
the  surface  of  a  solid  medium  and  distribute  it  through  the  drop. 
In  this  case  we  must  be  careful  to  take  very  little  of  the  material,  as 
the  smallest  quantity  will  contain  an  immense  number  of  bacteria, 
and  for  a  satisfactory  view  of  the  individual  cells  it  is  necessary  that 
they  be  well  separated  from  each  other,  in  some  parts  of  the  prepa- 
ration at  least,  and  not  massed  together. 

Where  the  object  is  to  make  a  cabinet  preparation  for  permanent 
preservation,  special  care  should  be  taken  to  distribute  the  bacteria 
uniformly  through  the  drop  of  water.  The  next  step  consists  in  eva- 
porating the  liquid  so  that  the  bacteria  may  remain  attached  to  the 
surface  of  the  glass  cover.  This  may  be  done  by  simple  exposure  to 
fche  air  or  by  the  application  of  gentle  heat.  When  the  bacteria  are 


•>(!  STAINING    METHODS. 

suspended  in  an  albuminous  medium  it  will  be  necessary,  after  the 
film  is  dry,  to  heat  the  preparation  sufficiently  to  coagulate  the  albu- 
rn an,  in  order  that  it  may  not  be  washed  off  in  the  subsequent  stain- 
ing process.  This  is  best  done,  in  accordance  with  Koch's  directions 
for  the  preparation  of  tuberculous  sputuzn,  by  passing  the  cover 
glass,  held  in  slender  forceps,  rather  quickly  through  the  flame  of  an 
alcohol  lamp  three  times  in  succession.  In  this  operation  it  must 
be  remembered  that  too  much  heat  will  destroy  the  preparation, 
while  too  little  will  fail  to  accomplish  the  object  in  view — coagu- 
lation of  the  albumen.  In  passing  the  cover  glass  through  the 
flame  the  smeared  side  is  to  be  held  upward.  The  time  required 
will  be  about  three  seconds  for  passing  it  three  times  as  directed  ; 
but  this  will  vary  according  to  the  intensity  of  the  flame,  and  some 
little  experience  is  necessary  in  order  to  obtain  tjie  best  results. 

The  operation  of  "  fixing,"  or  coagulating  the  albumen,  may  also 
be  effected  by  exposure  in  a  dry-air  oven,  heated  to  120°  to  130°  C., 
for  a  few  minutes  (two  to  ten  minutes),  as  directed  by  Ehrlich. 

Bacteria  simply  suspended  in  distilled  water  adhere  very  well  to 
the  cover  glass  when  treated  as  directed,  but  if  they  have  been  taken 
from  a  liquefied  gelatin  culture  the  film  is  very  apt  to  be  washed 
away  during  the  staining  process.  This  is  best  avoided  by  taking  as 
little  as  possible  of  the  gelatin  medium  and  suspending  the  bacteria 
to  be  examined  in  a  drop  of  water,  which  dilutes  the  gelatin  and 
washes  it  away  from  the  surface  of  the  cells. 

Smear  Preparations. — In  various  infectious  diseases  bacteria  are 
found  in  the  blood  and  tissues  of  the  body,  and  their  presence  may 
be  demonstrated  by  making  what  is  called  a  smear  preparation.  A 
little  drop  of  blood  may  be  spread  upon  the  thin  glass  cover,  or  it 
may  be  brought  in  contact  with  the  freshly  cut  surface  of  one  of  the 
vascular  organs,  as  the  liver  or  spleen.  It  is  especially  desirable  that 
the  material  used  for  such  a  preparation  be  small  in  amount  and  dis- 
tributed evenly  in  a  very  thin  layer.  In  Germany  it  is  the  custom, 
in  making  smear  preparations,  to  press  the  material  between  two  glass 
covers,  which  are  then  separated  by  sliding  them  apart,  thus  leaving 
a  thin  layer  upon  each.  This  answers  very  well,  but  the  writer  pre- 
fers to  spread  the  material  by  drawing  across  the  face  of  the  cover 
glass  the  end  of  a  well-ground  and  polished  glass  slide.  This  method 
is  especially  useful  for  spreading  blood  in  a  uniform  layer,  in  which 
the  corpuscles  are  evenly  distributed  and  retain  their  normal  form. 
A  very  small  drop  of  blood  is  placed  near  one  edge  of  the  cover  glass, 
which  is  placed  upon  a  smooth  surface  ;  the  glass  slide  is  held  at  a 
very  acute  angle  and  is  gently  drawn  across  the  cover  glass,  without 
any  pressure. 

Most  bacteriologists  make  their  preparations  upon  the  cover  glass, 


STAINING   METHODS.  27 

as  above  described,  but  the  writer  has  for  a  number  of  years  made 
his  mounts  of  bacteria  upon  the  glass  slide,  and  believes  that  this 
method  has  some  advantages  for  every-day  work.  The  thin  glass 
covers  required  when  a  preparation  is  to  be  examined  with  an  im- 
mersion objective  of  high  power,  are  easily  broken  and  often  dropped 
from  the  ringers  or  forceps.  When  the  material  to  be  examined  is 
spread  and  dried  directly  upon  the  glass  slide,  the  operation  is  at- 
tended with  less  difficulty  and  fewer  accidents  and  the  results  are 
quite  as  good.  In  this  case  the  slide  is  held  in  the  fingers  during  the 
various  steps  in  the  operation  of  distributing,  drying,  and  staining, 
while  the  thin  glass  cover  must  be  held  in  delicate  forceps. 

Contact  Preparations. — When  a  dry  and  clean  cover  glass  is 
brought  in  contact  with  a  colony  or  surface  culture  we  may  often 
obtain  a  very  pretty  preparation,  showing  the  bacteria  in  a  single 
layer,  and  preserving  the  arrangement,  as  regards  growth,  which 
characterizes  the  species.  Similar  preparations  may  sometimes  be 
obtained  from  the  surface  of  liquid  cultures,  when  the  bacteria  grow 
upon  the  surface  as  a  thin  film.  The  cover  glass  is  to  be  gently 
brought  into  contact  with  this  surface  growth,  which  adheres  to  it 
and  is  dried  and  stained  by  the  usual  methods. 

Staining  of  the  dried  film  is  quickly  effected  by  using  an  aqueous 
solution  of  one  of  the  aniline  colors  above  mentioned.  For  general 
use  the  writer  prefers  a  solution  of  f uchsin,  on  account  of  the  prompt- 
ness of  its  staining  action,  and  because,  in  preparations  for  permanent 
preservation,  it  is  not  as  likely  to  fade  as  methyleiie  blue  or  gentian 
violet.  It  is  also  a  better  color  than  blue  or  violet  in  case  a  photo- 
micrograph is  to  be  made  from  the  preparation. 

It  is  best  to  keep  on  hand  saturated  alcoholic  solutions  of  the 
staining  agents  named,  and  to  make  an  aqueous  solution  whenever 
required  by  the  addition  of  a  few  drops  to  a  little  water  in  a  watch 
glass  or  test  tube  ;  for  the  aqueous  solutions  do  not  keep  well  on  ac- 
count of  the  precipitation  of  the  dye  as  a  fine  powder,  which  ren- 
ders the  solution  opaque.  The  addition  of  ten  per  cent  of  alcohol 
to  the  aqueous  solution  will,  however,  prevent  this  precipitation ; 
but,  as  a  rule,  freshly  prepared  solutions  are  the  best.  These  should 
be  filtered  before  use.  We  may  place  a  few  drops  of  the  filtered 
solution  upon  the  dried  film  on  the  slide  or  cover  glass,  or  the  thin 
cover  may  be  floated  upon  a  little  of  the  solution  in  a  watch  glass. 
In  some  cases  it  is  best  to  use  heat  to  expedite  the  staining,  and  this 
ma}'  be  done  by  holding  the  slide  or  the  watch  glass  over  the  flame 
of  an  alcohol  lamp  until  steam  commences  to  be  given  off.  If  the 
heating  is  carried  too  far  the  preparation  is  likely  to  be  spoiled  by 
the  precipitation  of  the  staining  agent.  As  a  rule,  heating  will  not 
be  necessary,  and  when  an  aqueous  solution  of  f  uchsin  (one  part  to 


28  STAINING   METHODS. 

one  hundred  of  water)  is  used  most  bacteria  are  stained  within  a 
few  seconds  to  a  minute.  At  the  end  of  this  time  the  staining  solu- 
tion is  to  be  washed  away  by  means  of  a  gentle  stream  of  water,  or 
by  moving  the  cover  glass  about  in  a  vessel  containing  distilled 
water. 

Decolorization. — It  often  happens  that  the  albuminous  material 
associated  with  the  bacteria  which  we  propose  to  examine  is  stained 
so  deeply  as  to  obscure  the  view  of  these  ;  and,  generally,  we  will 
obtain  more  satisfactory  preparations  by  the  use  of  a  decolorizing 
agent,  by  which  the  background  is  cleared  up  and  the  outlines  of  the 
cells  more  clearly  defined.  The  agents  chiefly  used  for  this  purpose 
are  alcohol,  diluted  acids,  and  solution  of  iodine  with  potassium 
iodide  (Gram's  solution). 

Koch  recommends  a  solution  containing  sixty  parts  of  alcohol  to 
forty  parts  of  water.  The  cover  glass  is  to  be  quickly  passed 
through  this  solution  two  or  three  times.  Some  bacteriologists  pre- 
fer to  use  absolute  alcohol. 

Or  we  may  use  dilute  acetic  acid  (one-half  to  one  per  cent)  or 
very  dilute  hydrochloric  acid  (ten  drops  to  half  a  litre  of  water). 

For  decolorizing  preparations  containing  the  tubercle  bacillus 
strong  solutions  of  the  mineral  acids  are  employed  (one  part  of  ni- 
tric or  of  sulphuric  acid  to  three  parts  of  water). 

Gram's  solution  contains  one  part  of  iodine  and  two  parts  of 
potassic  iodide  in  three  hundred  parts  of  water.  Special  directions 
will  be  given  for  the  use  of  these  agents  when  we  give  an  account 
of  the  staining  methods  most  useful  for  the  various  pathogenic 
organisms. 

Double  Staining. — After  decolorizing  the  background  of  albu- 
minous material  we  may  again  stain  this  with  a  contrast  stain, 
such  as  eosin  or  vesuvin.  In  mounts  made  from  pure  cultures, 
either  liquid  or  solid,  a  single  stain,  for  the  bacteria  only,  is  all  that 
we  require,  and  our  aim  is  to  have  the  background  as  free  as  possi- 
ble from  any  material  which  would  obscure  the  view. 

After  staining,  decolorizing,  and  washing  the  preparation  the 
cover  glass  or  slide  is  again  dried  by  exposure  to  the  air  or  gentle 
heat,  and  is  then  ready  for  the  permanent  mounting  in  Canada  bal- 
sam. If  the  bacteria  have  been  stained  upon  the  slide,  a  small  drop 
of  balsam  dissolved  in  xylol  is  placed  in  the  middle  of  the  prepara- 
tion and  a  clean,  thin  glass  cover  applied. 

If  it  is  the  intention  to  make  the  microscopical  examination  with 
an  immersion  objective  of  high  power,  or  to  make  photomicro- 
graphs from  it,  only  the  thinnest  glass  covers  should  be  used — one- 
two-hundredths  of  an  inch  or  less. 

If  the  preparation  is  not  intended  for  permanent  preservation, 


STAINING   METHODS.  29 

the  examination  may  be  made  without  drying  the  surface  upon 
which  the  stained  bacteria  are  spread,  the  water  taking  the  place  of 
balsam  in  a  permanent  mount ;  or  we  may  dry  the  film  and  use  a 
drop  of  cedar  oil  between  the  slide  and  cover. 

While  simple  aqueous  solutions  of  the  aniline  colors,  when 
freshly  prepared,  will  promptly  stain  most  bacteria,  certain  agents 
may  be  added  to  these  which  aid  in  the  preservation  of  the  solution, 
or  which  act  as  mordants,  and  are  useful  in  special  cases. 

We  shall  only  give  here  a  few  of  the  standard  solutions  which 
are  most  frequently  employed  by  experienced  bacteriologists  : 

1.  Aniline- Gentian-Violet  (Ehrlich). 

Saturated  alcoholic  solution  of  gentian  violet,          .          .  5  cc. 

Aniline  water,       .......  100  cc. 

2.  Aniline- Methyl- Violet  (Ehrlich- Weigert). 

Saturated  alcoholic  solution  of  methyl  violet,          .          .11  cc. 
Absolute  alcohol,  ......  10  cc. 

Aniline  water,  .......          100  cc. 

Aniline  water  for  the  above  solutions  is  prepared  by  shaking  in  a 
test  tube  one  part  of  aniline  oil  with  twenty  parts  of  distilled  water, 
and,  after  allowing  it  to  stand  for  a  short  time,  filtering  the  saturated 
aqueous  solution  through  a  moistened  filter.  If  the  solution  is  not 
perfectly  transparent  it  should  be  filtered  a  second  time. 

3.   Carbol-Fuchsin  (Ziehl's  solution). 

Fuchsin,          .........      1  gm. 

Alcohol,  .  .          .          .          .          .          .         10  cc. 

Dissolve  and  add  100  cc.  of  a  fi\  3-per-cent  solution  of  carbolic  acid. 

4.  Alkaline  Blue  Solution  (Loffler's  solution). 

Saturated  solution  of  methyl ene  blue,         ...  30  cc. 

Solution  of  caustic  potash  of  1:10,000,  .  .  100  cc. 

These  solutions  keep  better  than  the  simple  aqueous  solutions, 
but  after  having  been  kept  for  a  time  they  are  likely  to  lose  their 
staining  power  as  a  result  of  the  precipitation  of  the  aniline  color. 

The  following  special  methods  of  staining  cover-glass  prepara- 
tions will  be  found  useful  in  certain  cases: 

Gram's  Method. — The  dried  film  upon  a  slide  or  cover  glass  is 
stained  with  an  aqueous  solution  of  methyl  violet  or  with  aniline- 
gentian-violet  solution  (No.  1);  it  is  then  placed  in  the  iodine  solution 
for  a  minute  or  two  (iodine  one  part,  potassic  iodide  two  parts,  water 


30  STAINING   METHODS. 

three  hundred  partis) ;  then  washed  in  alcohol,  dried,  and,  if  for  per- 
manent preservation,  mounted  in  balsam. 

METHODS  OF  STAINING  THE  TUBERCLE  BACILLUS.— Numerous 
methods  of  staining  the  tubercle  bacillus  in  sputum  dried  upon  a 
cover  glass  have  been  proposed,  but  we  shall  only  give  here  two  or 
three  of  the  most  approved  methods,  either  one  of  which  may  be 
relied  upon  for  satisfactory  results  if  carefully  followed. 

1.  The  Ehrlich-Weigert  Method. — Place  in  a  watch  glass  a  little 
of  the  aniline-methyl-violet  solution  (No.   2);  float  upon  the  surface 
of  this  the  cover  glass  with  the  dried  film  downward  ;  heat  over  a 
small  flame  until  it  begins  to  steam,  then  allow  it  to  stand  for  from 
two  to  five  minutes  ;  decolorize  in  a  tray  containing  one  part  of  nitric 
acid  to  three  parts  of  water — the  cover  glass,  held  in  forceps,  is  gently 
moved  about  in  the  decolorizing  solution  for  a  few  seconds.     It  is 
then  washed  off  in  sixty-per-<5ent  alcohol  to  remove  the  remaining 
blue  color — this  usually  takes  but  a  second  or  two — and  then  in  water. 
For  a  contrast  stain  a  saturated  aqueous  solution  of  vesuvin  may  be 
used,  a  few  drops  being  left  upon  the  cover  glass  for  five  minutes. 
The  stained   preparation  is  then  washed,  dried,    and  mounted  in 
balsam. 

2.  The  Ziehl-Neelson  Method. — Float  the  cover  glass  upon  the 
carbol-f uchsin  solution  (No.  3) ;  heat  gently  until  steam  commences 
to  rise — from  three  to  five  minutes'  time  will  usually  be  sufficient ; 
wash  off  in  water,  and  decolorize  in  nitric  or  sulphuric  acid,  twenty- 
five-per-cent  solution,  then  in  sixty-per-cent  alcohol  for  a  very  short 
time  to  remove  remaining  color  from  albuminous  background;  wash 
well  in  water  and  mount  in  Canada  balsam. 

3.  Friedldnder's  Method. — Spread  and  dry  the  sputum  upon 
the  slide ;  fix  by  passing  the  slide  three  times  through  the  flame  of 
an  alcohol  lamp  or  Bunsen  burner  ;  place  upon  the  dried  film  three  or 
four  drops  of  carbol-fuchsin  (No.  3);  heat  gently  over  a  flame  until 
steam  is  given  off  ;  wash  in  a  dish  of  distilled  water  ;  drain  off  excess 
of  water,  and  add  a  few  drops  of  the  following  decolorizing  solution : 

Acid,  nitric,  pure,          .  .  .  .  .  5  cc. 

Alcohol  (eighty  per  cent),  .  .  .  .to  100  cc. 

- — usually  the  preparation  will  be  decolorized  in  about  half  a  minute  ; 
wash  in  water  ;  add  a  few  drops  of  an  aqueous  solution  of  methylene 
blue  as  a  contrast  stain  ;  allow  the  stain  to  act  for  about  five  minutes, 
without  heating ;  wash  again  in  water,  dry,  and  mount  in  balsam, 
or  for  a  temporary  mount  use  a  drop  of  cedar  oil. 

4.  Gabbett's  Method. — This  is  a  slight  modification  only  of  a 
very  useful  method  recommended  by  B.  Frankel  in  1884.     The  con- 
trast stain  is  added  to  the  decolorizing  solution.     After  staining  with 


STAINING    METHODS.  31 

earbol-fuchsin  solution   (No.  />)   the   cover  glass  is  placed  for  one  or 
two  minutes  in  a  solution  containing: 

Sulphuric  acid  (t \venty-five-per-ceiit  solution),         .  .       100  cc. 

Melhylene  blue,       ......  2  gms. 

Wash,  dry,  and  mount  in  cedar  oil  or  balsam. 

METHODS  OF  STAINING  SPORES.— When  preparations  containing 
the  spores  of  bacilli  are  stained  by  any  of  the  methods  above  given, 
these  remain  unstained  and  appear  as  highly  refractive  bodies  in  the 
interior  of  the  rods  or  filaments  in  which  they  have  been  formed,  or 
scattered  about  in  the  field  if  they  have  been  set  free.  Owing  to 
the  contrast  with  the  stained  protoplasm  of  the  rod  or  spore-'bearing 
filament,  they  are  especially  well  seen  in  recent  cultures ;  while  in 
older  cultures  the  bacilli  often  do  not  stain  well,  or  are  entirely  dis- 
integrated and  spores  only  are  to  be  seen.  The  discovery  was  made 
at  about  the  same  time  by  Buchner  (1884)  and  by  Hueppe  that 
spores  may  be  stained  if  they  are  first  exposed  to  an  elevated  tem- 
perature for  some  time.  This  may  be  accomplished  by  placing  the 
slide  or  cover  glass,  upon  which  the  spore-containing  culture  has 
been  dried,  in  a  hot-air  oven  at  a  temperature  of  120°  C.  for  an 
hour;  or  a  higher  temperature  (180°  C.)  may  be  employed  for  a 
shorter  time  (fifteen  minutes)  ;  or  the  cover  glass  may  be  passed 
through  the  flame  of  an  alcohol  lamp  or  Bunsen  burner  eight  or  ten 
times,  instead  of  three  times  as  is  customary  when  the  object  in 
view  is  simply  to  coagulate  the  albumen  and  fix  the  film  upon  the 
cover  glass.  After  such  treatment  the  spores  may  be  stained  with 
an  aqueous  solution  of  one  of  the  basic  aniline  colors — fuchsin, 
methyl  violet,  etc. — but  the  bacilli  no  longer  take  the  stain  so  well. 

To  obtain  satisfactory  double-stained  preparations,  showing 
both  spores  and  bacilli,  a  different  method  is  employed. 

The  film  upon  the  cover  glass  is  passed  through  the  flame  three 
times,  as  heretofore  directed  ;  it  is  then  floated  upon  aniline-fuchsin 
solution  in  a  watch  glass,  and  this  is  heated  to  near  the  boiling  point 
for  an  hour — Neisser's  method.  The  aniline-fuchsin  solution  is 
prepared  by  shaking  an  excess  of  aniline  oil  in  a  test  tube  with  dis- 
tilled water,  filtering  the  saturated  solution  into  a  watch  glass,  and 
then  adding  a  few  drops  of  a  saturated  alcoholic  solution  of  fuchsin. 
After  this  prolonged  action  of  the  hot  staining  fluid  the  spores  of 
some  bacilli  are  deeply  stained,  while  others  do  not  take  the  stain  so 
well.  The  cover  glass  is  next  washed  in  water  and  then  placed  in 
a  decolorizing  solution  containing  twenty-five  parts  of  hydrochloric 
acid  to  seventy-five  parts  of  alcohol.  This  removes  the  stain  from 
the  bacilli,  but,  if  not  allowed  to  act  too  long,  leaves  the  spores  still 
stained.  The  preparation  is  next  stained  in  a  saturated  aqueous 


32  STAINING   METHODS. 

solution  of  methylene  blue;  and  if  the  operation  has  been  successfully 
carried  out  the  spores  will  be  stained  red  and  the  protoplasm  of  the 
bacilli  in  which  they  are  present  will  be  blue. 

Moller  has  (1891)  published  the  following  method  of  staining 
spores : 

The  cover-glass  preparation,  dried  in  the  air,  is  passed  three  times 
through  a  flame  or  placed  for  two  minutes  in  absolute  alcohol ;  it  is 
then  placed  in  chloroform  for  two  minutes  and  washed  in  water;  it 
is  now  immersed  in  a  five-per-cent  solution  of  chromic  acid  for  from 
half  a  minute  to  two  minutes  and  again  thoroughly  washed  in 
water;  next  a  solution  of  carbol-fuchsin  is  poured  upon  it  and  it 
is  heated  over  a  flame  until  it  commences  to  .boil,  for  sixty  seconds ; 
the  carbol-fuchsin  solution  is  then  poured  off  and  the  cover  glass  is 
immersed  in  a  five-per-cent  solution  of  sulphuric  acid  until  the 
film  is  decolorized,  after  which  it  is  again  thoroughly  washed  in 
water.  It  is  then  placed  for  thirty  seconds  in  an  aqueous  solution  of 
methylene  blue  or  of  malachite  green,  and  again  washed  in  water, 
after  which  the  preparation  should  be  dried  and  mounted  in  balsam. 
As  a  result  of  this  procedure  the  spores  are  stained  dark  red  and  the 
protoplasm  of  the  bacilli  blue  or  green. 

Fiocca  (1893)  claims  that  better  results  are  obtained  by  the  follow- 
ing method : 

About  twenty  cc.  of  a  ten-per-cent  ammonia  solution  is  placed  in  a 
watch  glass,  and  from  ten  to  twenty  drops  of  an  alkaline  solution  of 
an  aniline  color  is  added ;  heat  is  applied  until  steam  commences  to  be 
given  off,  when  the  cover  glass  is  placed  in  the  hot  solution  for  from 
three  to  fifteen  minutes.  The  cover  glass  is  then  quickly  washed  in 
a  twenty-per-cent  solution  of  nitric  or  sulphuric  acid  to  decolorize; 
then  it  should  be  thoroughly  washed  in  water,  after  which  it  may 
be  stained  with  a  contrast  color  by  the  use  of  an  aqueous  solution  of 
one  of  the  aniline  dyes — preferably  vesuvin,  malachite  green,  or 
safranin. 

METHODS  OF  STAINING  FLAGELLA. — Koch  first  succeeded  in  de- 
monstrating the  flagella  of  certain  bacilli  and  spirilla  by  staining 
them  with  an  aqueous  solution  of  haamatoxylon,  and  dilute  chromic 
acid  as  a  mordant.  Loftier  (1889)  has  succeeded  in  demonstrating, 
by  an  improved  staining  method,  the  presence  of  flagella  in  a  consider- 
able number  of  species  in  which  they  had  not  previously  been  seen, 
although  generally  suspected  to  be  present.  His  method  is  as  follows : 

Loffler\s  Method. — The  following  solution  is  used  as  a  mordant: 

No,  1. 

Solution  of  tannin  of  twenty  per  cent,          .  .  .10  cc. 

Saturated  (cold)  solution  of  ferrous  sulphate,    .  .  .     5  cc. 

Aqueous  or  alcoholic  solution  of  fuchsin,     .  .  .          1  cc. 

(Or  one  cubic  centimetre  alcoholic  solution  of  methyl  violet.) 


STAINING   METHODS.  33 

No.  2.. 

A  one-per-cent  solution  of  caustic  soda. 

No.  3. 

A  solution  of  sulphuric  acid  of  such  strength  that  one  cubic  centimetre 
is  exactly  neutralized  by  one  cubic  centimetre  of  the  soda  solution. 

According  to  Loffler,  solution  No.  1  is  just  right  for  staining  the 
flagellum  of  Spirillum  concentricum,  but  for  certain  other  bacteria  it 
is  necessary  to  add  to  this  some  of  No.  2  or  of  No.  3.  Thus,  for  the 
cholera  spirillum  from  half  a  drop  to  a  drop  of  the  acid  solution  is 
added  to  sixteen  cubic  centimetres  of  No.  1.  For  the  bacillus  of 
typhoid  fever  one  cubic  centimetre  of  No.  2  is  added  to  sixteen  cubic 
centimetres  of  No.  1.  Bacillus  subtilis  requires  twenty-eight  to 
thirty  drops  of  No.  2;  the  bacillus  of  malignant  cedema  thirty-six  to 
thirty-seven  drops,  etc. 

This  method  has  not  been  very  successful  in  the  hands  of  other 
bacteriologists,  and  improvements  in  the  technique  have  been  made 
since  it  was  first  published.  Van  Ermengem  (1893)  points  out  the 
fact  that  a  principal  condition  of  success  is  that  the  cover  glasses  shall 
be  absolutely  clean.  He  boils  them  in  a  mixture  composed  of  potas- 
sium bichromate,  sixty  grammes;  concentrated  sulphuric  acid,  sixty 
grammes ;  water,  one  hundred  grammes.  After  coming  from  this  they 
are  thoroughly  washed  in  water,  then  in  absolute  alcohol,  and  then 
dried  in  an  upright  position  under  a  bell-jar.  Recent  agar  cultures 
(ten  to  eighteen  hours)  are  preferred,  and  the  suspension  in  water 
should  be  very  much  diluted  so  that  in  the  cover-glass  preparation 
the  bacteria  are  well  isolated.  The  cover  glass,  held  between  the 
fingers,  is  passed  three  times  through  a  flame.  A  drop  of  the  follow- 
ing solution  is  then  placed  upon  it :  Osmic  acid  two-per-cent  solution, 
one  part ;  solution  of  tannin  (ten  to  twenty -five  per  cent)  two  parts. 
This  is  allowed  to  act  for  about  five  minutes  at  a  temperature  of  50° 
to  60°  C. — or  half  an  hour  at  the  room  temperature.  After  careful 
wrashing  with  water  and  alcohol  the  cover  glass  is  immersed  for  a 
few  seconds  in  a  bath  containing  one-quarter  to  one-half  per  cent  of 
nitrate  of  silver.  Then  without  washing  it  is  placed  for  a  short 
time  in  the  following:  Gallic  acid,  five  grammes;  tannin,  three 
grammes;  fused  potassium  acetate,  ten  grammes;  distilled  water, 
three  hundred  and  fifty  grammes.  It  is  then  returned  to  the  silver 
bath  and  kept  there,  with  constant  movement  of  the  bath,  until  this 
commences  to  turn  black.  It  is  then  thoroughly  washed  in  water, 
dried,  and  mounted  in  balsam. 

Pitfield  (1895)  has  devised  a  much  simpler  method  which,  as 
modified  by  Muir,  is  as  follows : 

"  Prepare  the  following  solutions  : 
3 


34  STAINING     METHODS. 


A. — THE  MORDANT. 

Tannic  acid,  ten-per-cent  aqueous  solution,  filtered,  .         10  c.c. 

Corrosive  sublimate,  saturated  aqueous  solution,  .                  5  c.c. 

Alum,  saturated  aqueous  solution,    .             .             .  .           5  c.c. 

Carbol-fuchsin  solution,1               .            .            .  .            .     5  c.c. 

''Mix  thoroughly.  A  precipitate  forms,  which  must  be  allowed  to  de- 
posit, either  by  centrifugalizing  or  simply  by  allowing  to  stand.  Remove 
the  clear  fluid  with  a  pipette  and  transfer  to  a  clean  bottle.  The  mordant 
keeps  well  for  one  or  two  weeks. 

B.— THE  STAIN. 

Alum,  saturated  watery  solution,      .  .  .  .10  c.c. 

Gentian  violet,  saturated  alcoholic  solution,     .  .  2  c.c. 

"  The  stain  should  not  be  more  than  two  or  three  days  old  when  used.  It 
may  be  substituted  in  the  mordant  in  place  of  the  carbol  fuchsin.  The  film 
having  been  prepared  as  above  described,  pour  over  it  as  much  of  the  mor- 
dant as  the  cover  glass  will  hold.  Heat  gently  over  a  flame  till  steam  begins 
to  rise,  allow  to  steam  for  about  a  minute,  and  then  wash  well  in  a  stream 
of  running  water  for  about  two  minutes.  Then  dry  carefully  over  the  flame, 
and  when  thoroughly  dry  pour  on  some  of  the  stain.  Heat  as  before,  allow- 
ing to  steam  for  about  a  minute,  wash  well  in  water,  dry  and  mount  in  a 
drop  of  xylol  balsam ?'  (Muir  and  Ritchie). 

METHODS  OF  STAINING  BACTERIA  IN  TISSUES. — The  solutions  re- 
commended for  staining  cover-glass  preparations  are  also  used  in 
staining  bacteria  in  thin  sections  of  the  various  organs,  in  which 
they  are  found  in  certain  infectious  diseases;  but,  in  general,  a 
longer  time  is  required  to  stain  sections,  and  it  is  best  not  to  hasten 
the  process  by  the  use  of  heat.  To  obtain  good  thin  sections,  the 
material,  cut  in  small  cubes,  must  bs  very  thoroughly  hardened  in 
absolute  alcohol.  The  piece  selected  for  cutting  may  be  attached  to 
a  cork  by  the  use  of  melted  glycerin  jelly,  which  is  hardened  by 
placing  the  cork  and  attached  piece  of  tissue  in  alcohol.  This  an- 
swers for  well-hardened  pieces  of  liver,  kidney,  etc. ,  but  the  hollow 
viscera  and  tissues  of  loose  structure  will  require  embedding  in 
paraffin  or  celloidin.  Any  well-made  sledge  microtome  will  answer 
for  cutting  the  sections,  if  the  knife  is  properly  sharpened.  The  sec- 
tions should,  of  course,  be  cut  under  alcohol,  and  they  can  scarcely 
be  too  thin  when  the  object  is  to  demonstrate  the  presence  or  ab- 
sence of  bacteria.  Very  thin  sections  may  be  cut  dry  by  embedding 
in  paraffin  having  a  melting  point  of  50°  C.  In  this  case  the  knife 
is  set  at  a  right  angle  to  the  material  to  be  cut,  and  the  sections 
are  spread  out  upon  and  attached  to  the  glass  slide  for  staining. 

One  of  the  most  useful  solutions  for  staining  tissues  is  Loffler's 
alkaline  solution  of  methylene  blue  (No.  4).  A  freshly-prepared  so- 

1  Basic  fuchsin,  1  part;  absolute  alcohol,  10  parts;  solution  of  carbolic  acid 
(1  :  20),  100  parts. 


STAINING    METHODS.  35 

lution  will  stain  sections  in  four  or  five  minutes.  Superfluous  color 
is  removed  by  immersing  the  sections  in  diluted  alcohol  or  in  a  one- 
half -per-cent  solution  of  acetic  acid  for  a  few  seconds.  The  sections 
are  dehydrated  in  absolute  alcohol,  cleared  up  with  oil  of  cedar,  and 
mounted  in  a  drop  of  cedar  oil  for  examination,  or  in  balsam  if 
they  are  to  be  preserved. 

Grain's  method  may  be  used  as  directed  for  cover-glass  prepara- 
tions, the  sections  being  first  stained  in  aniline-gentian-violet  solu- 
tion (No.  1),  then  washed  in  water,  or  in  aniline  water  as  recently 
(1892)  recommended  by  Botkin,  then  decolorized  in  the  iodine  solu- 
tion (see  page  29).  The  sections  when  decolorized  are  again  washed 
in  water,  dehydrated  in  absolute  alcohol,  cleared  in  cedar  oil,  and 
mounted  in  balsam. 

Weigert's  Method. — This  is  a  modification  of  Gram's  method  in 
which  the  sections  are  dehydrated  by  the  use  of  aniline  oil.  The 
stained  section,  after  having  been  washed,  is  transferred  to  a  clean 
glass  slide,  the  excess  of  water  is  removed  by  the  use  of  filtering 
paper,  and  the  iodine  solution  is  placed  upon  it  in  sufficient  quantity 
to  cover  the  entire  section.  When  sufficiently  decolorized  this  is  re- 
moved in  the  same  way.  The  section  is  then  dehydrated  by  placing 
a  few  drops  of  aniline  oil  upon  it,  removing  this  with  filtering  paper, 
and  repeating  the  operation  once  or  twice.  The  aniline  oil  must 
then  be  completely  removed  by  the  use  of  xylol,  after  which  the  sec- 
tion is  mounted  in  balsam. 

Kiihne's  Method. — The  object  of  this  method  is  to  prevent  the  removal 
of  the  color  from  stained  bacteria  in  sections  during  the  treatment  which 
such  sections  usually  receive  before  they  are  ready  for  mounting — i.e., 
during  the  washing  and  dehydrating  processes  usually  employed.  For 
staining,  Kiihne  prefers  a  methylene-blue  solution  prepared  as  follows: 
Methylene  blue,  1.5  parts;  absolute  alcohol,  ten  par-ts  ;  triturate  in  a  watch 
glass  and  add  gradually  one  hundred  parts  of  a  solution  of  carbolic  acid 
containing  five  parts  in  one  hundred  of  water.  The  section  is  placed  in  this 
solution  for  about  half  an  hour,  then  washed  in  water  and  decolorized  in  a 
weak  solution  of  hydrochloric  acid — ten  drops  to  five  hundred  grammes  of 
water.  This  part  of  the  operation  must  be  conducted  very  carefully,  and 
usually  thin  sections  will  only  require  to  be  dipped  in  the  acid  solution  for  an 
instant,  after  which  they  must  be  at  once  immersed  in  a  solution  of  lithium 
— eight  drops  of  a  saturated  solution  of  carbonate  of  lithium  in  ten  grammes 
of  water.  They  are  then  allowed  to  remain  in  a  bath  of  distilled  water  for 
a  few  minutes,  after  which  they  are  dipped  into  absolute  alcohol,  which 
Kiihne  colors  by  the  addition  of  methyl  en  e  blue.  The  sections  are  then 
placed  in  aniline  oil  which  contains  a  little  methylene  blue  in  solution, 
where  they  are  dehydrated  without  the  color  being  extracted  from  the  stained 
bacteria  present.  The  aniline-oil  blue  solution  is  prepared  by  adding  an  ex- 
cess of  dry  methylene  blue  to  a  small  quantity  of  clarified  aniline  oil.  The 
undissolved  pigment  settles  to  the  bottom,  and  a  few  drops  of  the  colored 
solution  are  added  to  a  little  aniline  oil  in  a  watch  glass  to  make  the  colored 
dehydrating  bath.  The  section  is  next  washed  out  in  pure  aniline  oil — not 
colored — after  which  every  trace  of  aniline  oil  is  to  be  removed  by  the  use 
of  xylol.  The  section  is  cleared  up  in  turpentine  and  mounted  in  balsam. 


36  STAINING   METHODS. 

Ziehl-Neelson  Method,  for  the  tubercle  bacillus  in  tissues.—- 
Leave  the  sections  for  fifteen  minutes  in  carbol-fuchsin  solution 
(No.  3) ;  decolorize  in  sulphuric  or  nitric  acid,  twenty-five-per-cent 
solution;  wash  in  sixty -per-cent  alcohol;  place  in  a  saturated  aque« 
ous  solution  of  methylene  blue  for  contrast  stain;  wash,  dehydrate, 
and  mount  in  balsam. 

The  following-  method  of  staining1  sections  for  the  purpose  of  demon- 
strating bacteria  present  in.  the  tissues  is  recommended  by  Pregl  (1891)  as  a 
substitute  for  the  method  of  Klihne.  The  results  are  said  to  be  excellent, 
and  it  is  much  simpler  and  more  expeditious. 

The  sections  are  made  from  tissues  embedded  in  paraffin,  and  are  attached 
to  clean  glass  slides  with  albumen-glycerin.  Or  they  may  be  attached  to  a 
cover  glass  by  the  following  method  when  not  embedded  in  paraffin :  The 
sections,  completely  dehydrated,  are  taken  out  of  absolute  alcohol  on  a  thin 
glass  cover,  upon,  which  they  are  extended  ;  a  piece  of  filter  paper  is  applied 
to  the  side  of  the  cover  glass  to  absorb  the  alcohol,  and  before  the  section  is 
completely  dry  a  drop  of  acetoii-celloidin  solution  is  placed  upon  it  by  means 
of  a  glass  rod.  The  cover  glass  is  now  moved  about  in  the  air  to  promote 
rapid  evaporation  of  the  alcohol,  and  is  then  placed  in  water.  The  section 
now  remains  attached  to  the  cover  glass  during  subsequent  manipulations. 
The  aceto)i-celloidin  solution  referred  to  is  prepared  by  adding  celloidin  in 
small,  dry  pieces  to  acetoii  until  a  concentrated  solution  is  obtained.  A 
large  drop  of  this  added  to  five  cubic  centimetres  of  absolute  alcohol  makes 
a  suitable  solution  for  use.  This  must  be  kept  in  a  glass-stoppered  bottle,  and 
will  require  to  be  frequently  renewed,  as  it  is  not  suitable  for  use  after  hav- 
ing absorbed  moisture  from  the  air.  The  acetoii  as  obtained  from  dealers 
contains  considerable  water  and  must  be  dehydrated  by  adding  to  it  red-hot 
sulphate  of  copper. 

The  sections,  attached  to  a  slide  or  cover  glass  by  one  of  the  methods 
mentioned,  are  stained  with  Kuhne's  carbol-methylene-blue  solution,  which 
is  dropped  upon  them  from  a  pipette.  Usually  they  will  be  sufficiently 
stained  at  the  end  of  half  a  minute  to  a  minute,  but  in  some  cases  a  longer 
time  and  the  application  of  heat  will  be  desirable.  They  are  then  washed  in 
water  and  immediately  placed  in  fifty-per-cent  alcohol,  where  they  remain 
until  the  sections  have  a  pale-blue  color  with  a  greenish  tinge.  They  are 
now  completely  dehydrated  in  absolute  alcohol  and  subsequently  cleared  up 
in  xylol. 

STAINING  SECTIONS  OF  GELATIN  STICK  CULTURES. — Fischl,  Weigert, 
and  Neisser  have  given  an  account  of  methods  for  staining  stick  cultures  in 
gelatin  of  non-liquefying  bacteria.  The  object  of  this  is  to  show  the  mode 
of  growth  and  the  association  of  individual  cells  in  undisturbed  cultures. 
Neisser  gives  the  following  directions  :  The  gelatin  cultures  are  inoculated, 
by  several  punctures,  with  the  microorganism  to  be  studied.  When  the 
development  is  deemed  sufficient  the  cylinder  of  gelatin  is  removed  from  the 
test  tube  by  gently  warming  its  walls.  It  is  then  placed  for  several  days- 
one  to  eight,  according  to  its  size  and  thickness — in  a  one-per-cent  solution  of 
bichromate  of  potassium.  While  in  this  solution  it  must  be  exposed  to  the 
light,  which  causes  a  change  in  the  gelatin,  rendering  it  insoluble.  The 
gelatin  cylinder  is  thoroughly  washed  and  then  hardened  in  alcohol,  first  of 
seventy  per  cent,  and  then  of  ninety-six  per  cent.  It  is  then  cut  into  suit- 
able pieces,  and  these  are  attached  to  a  cork  in  the  usual  manner  and  placed 
for  twenty-four  hours  in  absolute  alcohol.  Thin  sections  may  now  be  made 
with  a  microtome,  and  these  are  attached  to  a  glass  slide  and  stained  by 
Gram's  or  Weigert's  method  or  by  the  use  of  Loffler's  solution  (No.  4).  The 
decolorization  should  be  effected  by  the  use  of  alcohol  and  not  with  an  acid 
solution.  When  Gram's  method  'is  used  decolorize  by  the  alternate  use  of 
alcohol  and  oil  of  cloves.  Clear  the  preparation  with  oil  of  bergamot. 


V. 
CULTURE  MEDIA. 

To  obtain  a  satisfactory  knowledge  of  the  biological  characters 
of  the  different  species  of  bacteria,  it  is  necessary  to  isolate  them  in 
"  pure  cultures  "  and  to  study  their  growth  in  various  culture  media. 
By  a  pure  culture  we  mean  a  cultivation  containing  a  single  species 
only ;  and  to  be  absolutely  sure  that  we  have  a  pure  culture  it  is 
desirable  that  all  of  the  bacteria  in  a  culture  shall  be  the  progeny  of 
a  single  cell.  The  methods  of  obtaining  pure  cultures  will  be  given 
later.  At  present  we  propose  to  give  an  account  of  the  various  cul- 
ture media  commonly  employed  by  bacteriologists,  and  the  methods 
of  preparing  them  for  use. 

By  a  natural  culture  medium  we  mean  one  which,  as  obtained  in 
nature,  contains  the  necessary  pabulum  for  the  development  of  one 
or  more  species  of  bacteria.  An  artificial  culture  medium  is  one 
which  is  prepared  artificially  by  adding  nutritive  material  to  water. 
A  sterile  medium  is  one  which  does  not  contain  any  living  micro- 
organisms. We  may  obtain  natural  media  in  a  sterile  condition,  but 
artificial  media  require  sterilization,  as  they  are  infallibly  contami- 
nated with  living  "  germs  "  from  the  atmosphere  during  the  process 
of  preparing  them.  Sterilization  is  usually  effected  by  heat.  For- 
ceps, glass  tubes,  etc. ,  may  be  sterilized  by  passing  them  through 
the  flame  of  an  alcohol  lamp  or  Bunsen  burner. 

NATURAL  CULTURE  MEDIA. — The  most  important  natural  cul- 
ture medium  is  blood  serum,  which  may  be  obtained  from  one  of 
the  lower  animals — preferably  from  oxen  or  calves.  This  is  to  be 
collected  in  a  sterilized  jar,  with  every  precaution  to  insure  cleanli- 
ness, at  the  moment  of  slaughtering  the  animal.  Or  the  blood  of  a 
calf,  sheep,  or  dog  may  be  collected  at  the  laboratory  by  a  carefully 
conducted  operation,  in  which  the  femoral  or  carotid  artery  is  con- 
nected with  a  sterilized  glass  tube  leading  into  a  sterilized  receptacle, 
such  as  a  Woulf's  bottle,  into  one  neck  of  which  a  cotton  plug  has 
been  placed  to  permit  the  air  to  escape  as  the  bottle  fills  with 
blood  through  a  tube  which  is  secured  in  the  other  neck.  When 
blood  is  passed  directly  from  an  artery  into  a  sterilized  receptacle 
the  serum  will  not  subsequently  require  sterilization.  The  writer  is  in 


MEDIA. 


the  habit  of  collecting  it  in  this  way,  and,  after  the  serum  has  sepa- 
rated, of  drawing  it  off  in  little  flasks  having  a  long  neck,  as  shown 
in  Fig.  14.  The  neck  of  the  flask,  previously  sterilized  by  heat,  is 
slipped  into  the  Woulf's  bottle  beside  the  cotton  plug,  the  bulb  (a) 
having  been  previously  gently  heated  to  expand  the  contained  air. 
As  the  heated  air  cools  a  partial  vacuum  is  formed  and  the  clear 
serum  mounts  into  the  little  flask.  One  after  another  is  filled  in 
this  way,  and  each  one  is  hermetically  sealed  in  the  flame  of  a  lamp 


-a 


\ 


Fio.  14. 


Fio.  15. 


FIG.  18. 


as  soon  as  it  is  withdrawn.  The  sterile  blood  serum  may  be  pre- 
served indefinitely  in  this  way,  and  may  be  used  as  a  liquid  culture 
medium  in  the  little  flask,  or  it  may  be  transferred  to  a  test  tube 
and  solidified  by  heat  whenever  a  solid  blood-serum  medium  is  re- 
quired. The  advantage  of  preserving  blood  serum  and  other  liquid 
media  in  these  little  flasks  is  in  the  fact  that  they  may  be  preserved 
indefinitely  without  becoming  contaminated  or  drying  up,  and  that 
they  are  easily  transported,  while  a  liquid  medium  in  a  test  tube 
must  be  kept  upright.  The  contents  of  one  of  these  flasks  are  readily 


CULTURE   MEDIA.  39 

transferred  to  a  test  tube  by  breaking  off  the  sealed  extremity  with 
sterile  forceps  and  slipping  it  past  the  cotton  plug,  which  must  be 
partly  withdrawn  for  the  purpose.  Upon  applying  gentle  heat  to 
the  bulb  its  contents  are  forced  out  into  the  test  tube  (Fig.  15). 
Blood  serum  which  is  collected  without  these  special  precautions 
will  require  sterilization  by  heat,  for  which  directions  will  be  given 
later. 

To  obtain  the  clear  serum  from  blood  collected  as  above  directed, 
the  jars  containing  it  are  set  aside  in  a  cool  place  in  order  that  a  firm 
clot  may  form,  care  being  taken  not  to  shake  them.  After  the  clot 
has  formed  they  may  be  transported  to  the  laboratory,  where  they 
are  placed  in  an  ice  box  or  in  a  cool  cellar  for  from  twenty-four  to 
forty-eight  hours.  By  this  time  the  serum  has  separated  from  the 
clot,  and  it  may  be  transferred  to  sterilized  test  tubes  by  means  of  a 
suction  pipatte  (Fig.  16),  or  may  be  distributed  in  little  flasks  as 
above  directed 

31  ilk  is  largely  used  as  a  culture  medium,  and  is  especially  useful 
in  studying  the  biological  characters  of  various  microorganisms,  as 
shown  by  their  causing  coagulation  of  the  casein,  or  otherwise  ;  or 
an  acid  or  alkaline  reaction  of  the  liquid  ;  or  peptonization  of  the 
precipitated  casein,  etc.  In  the  udder  of  healthy  cows  milk  is  quite 
sterile,  and  by  proper  precautions  it  may  be  drawn  into  sterilized 
flasks  without  any  contamination  and  kept  indefinitely  without  un- 
dergoing coagulation  or  any  other  change.  But  in  practice  it 
is  easier  to  sterilize  it  in  test  tubes  or  small  flasks  by  the  use  of 
heat  than  to  obtain  it  in  a  sterile  condition  from  the  udder  of  the 
cow. 

[TrmeJaas  been  used  to  some  extent  as  a  culture  medium,  and 
many  bacteria  multiply  in  it  abundantly,  although,  on  account  of  its 
acid  reaction,  other  species  fail  to  grow  in  it.  As  contained  in  the 
healthy  bladder  it  is  sterile,  but  the  mucous  membrane  of  the  mea- 
tus  urinarius  always  contains  numerous  bacteria  upon  its  surface,  and 
some  of  these  are  sure  to  be  carried  away  with  the  current  when 
urine  is  passed. 

A  culture  fluid  which  the  writer  has  found  extremely  useful,  in 
tropical  countries  where  it  is  to  be  obtained,  is  the  transparent  fluid 
contained  in  the  interior  of  unripe  cocoanuts — called  agua  coco  by 
the  Spaniards.  In  countries  where  the  cocoanut  is  indigenous  this 
cocoanut  water  is  largely  used  as  a  refreshing  drink.  It  contains 
about  four  per  cent  of  glucose  in  solution,  together  with  some  vege- 
table albumen  and  salts.  Some  microorganisms  multiply  in  it  with- 
out appropriating  the  glucose,  while  others  split  this  up,  producing 
an  abundant  evolution  of  carbon  dioxide  and  giving  to  the  fluid 
a  very  acid  reaction.  The  following  are  the  results  of  an  analysis 


40  CULTURE   MEDIA. 

made  for  me  by  Dr.  L.  L.  Van  Slyke  in  the  chemical  laboratory  of 
Johns  Hopkins  University  :  The  weight  of  the  fluid  obtained  from 
six  nuts  averaged  339.1  grammes.  The  specific  gravity  averaged 
1.02285.  The  amount  of  water  averaged  95  percent;  the  amount 
of  inorganic  ash,  0.618  per  cent;  the  amount  of  glucose,  3.97  per 
cent ;  the  amount  of  fat,  0.119  per  cent ;  the  amount  of  albuminoids, 
0.133  percent. 

As  this  fluid  is  contained  in  a  germ-proof  receptacle,  no  steriliza- 
tion is  required  when  it  is  drawn  off  with  proper  precautions  in  the 
little  flasks  heretofore  described. 

Hydrocele  fluid  has  been  used  as  a  culture  medium,  and  many 
bacteria  multiply  in  it  abundantly. 

Other  natural  culture  media  are  found  in  animal  and  vegetable 
substances,  which  are  used,  either  cooked  or  raw,  as  solid  sub- 
strata upon  which  bacteria  may  be  cultivated.  One  of  the  most  use- 
ful of  these  is  the  potato,  which  is  a  favorable  medium  for  the  de- 
velopment of  numerous  species,  and  upon  which  (cooked)  many  of 
them  present  characters  of  growth  which  are  so  distinctive  as  to  aid 
greatly  in  the  differentiation  of  species. 

Other  tubers,  roots,  or  fruits  may  also  be  used  as  solid  media,  or 
their  juices  extracted  and  employed  as  liquid  media.  Cooked  fish 
and  meats  of  various  kinds  are  also  suitable  media  for  certain  spe- 
cies— e.g.,  the  phosphorescent  bacteria  grow  very  well  upon  the  sur- 
face of  boiled  fish,  and  in  a  dark  room  give  off  a  bright,  phosphores- 
cent light. 

Eggs,  sterilized  "by  boiling,  have  been  used  by  some  bacteriolo- 
gists, especially  for  the  cultivation  of  anaerobic  species. 

ARTIFICIAL  CULTURE  MEDIA. — A  great  variety  of  liquid  media 
have  been  employed  by  bacteriologists,  the  most  useful  of  which  are 
infusions  of  beef  or  mutton,  with  the  addition  of  a  little  peptone. 
But  Pasteur  has  shown  that  some  species  of  bacteria  will  grow  in  a 
medium  which  does  not  contain  any  albuminous  material,  nitrogen 
being  obtained  from  salts  containing  ammonia. 

Pasteur's  solution,  which  is  rarely  used  at  present,  contains  : 
Distilled  water,  one  hundred  parts  ;  cane  sugar,  ten  parts  ;  tartrate 
of  ammonia,  one  part,  with  the  addition  of  the  ashes  from  one 
gramme  of  yeast. 

Cohn  modified  this  by  leaving  out  the  cane  sugar,  which  favors 
the  development  of  moulds.  These  fluids  are  not,  however,  in- 
tended for  general  use  in  the  cultivation  of  bacteria,  but  to  demon- 
strate certain  facts  relating  to  their  physiology. 

Infusions  of  meat,  or  "  flesh  water,"  are  made  by  chopping  fine 
lean  beef  or  mutton  (one  pound)  and  covering  it  with  water  (one 
litre).  This  is  placed  in  an  ice  chest  for  twenty-four  hours,  and  the 


CULTURE   MEDIA.  41 

aqueous  extract  is  then  obtained  by  filtration  through  muslin  by 
pressure.  This  extract  is  cooked,  filtered,  and  carefully  neutralized 
by  the  addition  of  a  solution  of  carbonate  of  sodium,  which  is  added 
drop  by  drop.  Usually  we  add  to  this  one-half  per  cent  of  chloride 
of  sodium.  The  addition  of  ten  grammes  of  peptone  to  a  litre  of 
this  meat  infusion  constitutes  the  flesh-peptone  solution  which  is 
largely  used  in  the  preparation  of  solid  culture  media,  to  be  described 
hereafter. 

The  addition  of  five  per  cent  of  glycerin  to  the  above  infusion 
makes  a  useful  liquid  medium  for  the  cultivation  of  the  tubercle  ba- 
cillus (Roux  and  Xocard).  The  liquid  should  be  again  neutralized 
after  adding  the  glycerin,  which  commonly  has  an  acid  reaction. 

Dunham's  Peptone  Solution. — This  is  used  principally  for  de- 
termining whether  bacteria  under  investigation  are  capable  of  pro- 
ducing indol.  One  part  of  pure  dried  peptone  is  added  to  100  parts 
of  distilled  water,  and  to  this  is  added  one-half  per  cent  of  sodium 
chloride.  The  addition  of  rosalic  acid  to  this  solution  affords  a 
means  of  determining  whether  bacteria  cultivated  in  it  produce  an 
acid  or  an  alkaline  reaction  of  the  medium.  The  pale  rose  color  im- 
parted to  the  peptone  solution  by  the  addition  of  rosalic  acid  becomes 
more  intense  when  the  solution  becomes  alkaline,  and  it  fades  out  en- 
tirely when  it  becomes  acid.  To  obtain  this  reaction  add  2  parts 
of  the  following  solution  to  100  parts  of  Dunham's  peptone  solution: 
rosalic  acid  (corralline),  2  parts;  alcohol  (eighty  per  cent),  100  parts. 

Bouillon  is  made  by  cooking  the  chopped  meat — one  pound  in  a 
litre  of  water — for  about  half  an  hour  in  a  large  glass  flask  or  an 
enamelled  iron  kettle.  The  filtered  bouillon  is  then  carefully  neu- 
tralized with  sodium  carbonate,  and  again  boiled  for  an  hour  to  pre- 
cipitate all  coagulable  albuminoids.  It  is  again  filtered  and  dis- 
tributed in  test  tubes  or  small  flasks,  in  which  it  is  subsequently 
sterilized.  For  certain  pathogenic  bacteria  a  bouillon  made  from  the 
flesh  of  a  fowl  or  of  a  rabbit  is  preferable  to  beef  bouillon. 

Flesh  infusion  may  also  be  made  from  one  of  the  standard  beef 
extracts,  such  as  Liebig's  (five  grammes  to  a  litre  of  water). 

Various  vegetable  infusions  may  also  be  used  as  culture  media, 
such  as  yeast  water,  potato  water,  infusion  of  hay,  of  barley,  or  of 
wheat,  of  dried  fruits,  beer  wort,  etc. 

SOLID  CULTURE  MEDIA.— The  introduction  of  solid  culture 
media,  and  especially  the  use  of  gelatin  and  agar-agar,  as  first 
recommended  by  Koch  (1881),  for  the  isolation  and  differentiation  of 
species,  was  a  most  important  advance  in  bacteriological  technology. 
We  are  concerned  here  only  with  the  composition  and  preparation 
of  these  media. 

Flesh-Pept  one-Gelatin. — This  is  made  by  adding  ten  per  cent 


42 


CULTURE    MEDIA. 


of  the  best  French  gelatin  to  the  flesh-peptone  solution  above  de- 
scribed. This  is  the  standard  gelatin  medium,  but  more  or  less 
gelatin  may  be  added  to  serve  a  special  purpose.  Thus,  in  Havana 
during  the  summer  months  the  writer  used  a  medium  containing 
twenty  per  cent  of  gelatin,  because  when  but  ten  per  cent  was  used 
the  gelatin  was  liquefied  by  the  normal  temperature  of  the  atmo- 
sphere. Ten-per-cent  gelatin,  of  good  quality  and  carefully  pre- 
pared, will  stand  a  temperature  of  20°  to  22°  C.  (68°  to  71.6°  F.) 
without  melting.  When  twenty  per  cent  of  gelatin  is  used  the 
melting  point  is  about  8°  C.  higher.  It  must  be  remembered  that 
exposure  to  a  boiling  temperature  reduces  the  melting  point  of  gela- 
tin. It  is  therefore  desirable  to  accomplish  the  operations  of  cook- 


FIG.  17. 


ing  and  sterilizing  in  as  short  a  time  as  is  practicable.  The  French 
gelatin  used  comes  in  thin  sheets  ;  this  is  broken  up  and  added  to 
the  flesh-peptone  solution. 

Usually  we  prepare  a  litre  of  nutrient  gelatin  at  one  time,  and  for 
this  quantity  one  hundred  grammes  of  gelatin  will  be  required  for  the 
standard  preparation  (ten  per  cent).  It  is  well  to  allow  it  to  soak  for 
a  time  in  the  liquid  before  applying  heat  for  the  purpose  of  dissolving 
it.  Then  apply  gentle  heat  until  it  is  completely  dissolved.  The  gela- 
tin of  commerce  usually  has  an  acid  reaction,  and  it  will  be  necessary 
to  carefully  neutralize  the  medium  after  it  has  been  added.  A  slightly 
alkaline  reaction  is  usually  no  disadvantage,  but  certain  pathogenic 
bacteria  will  not  grow  when  there  is  a  trace  of  acid  present.  The 
next  step  consists  in  clarifying  the  nutrient  medium.  It  is  allowed 


CULTURE    MEDIA.  43 

to  cool  to  about  50°  C.,  and  an  egg,  previously  broken  into  one 
hundred  grammes  of  water,  is  gradually  added  while  stirring  the 
liquid  with  a  glass  rod.  A  whole  egg  is  used  for  a  litre  of  the  solu- 
tion. Heat  is  again  applied  and  the  solution  is  kept  at  the  boiling 
point  for  about  ten  minutes,  during  which  time  the  egg  albumen  is 
precipitated  and  carries  down  with  it  all  insoluble  particles,  which 
without  this  clarifying  process  would  have  interfered  with  the  trans- 
parency of  the  medium,  even  when  carefully  filtered.  The  hot 
solution  is  then  filtered.  A  hot- water  funnel  (Fig.  17)  is  usually 
employed,  as  the  gelatin  solution  does  not  pass  through  filtering 
paper  very  rapidly,  and  when  cooled  to  near  the  point  of  solidifying 
ceases  to  pass. 

The  advantages  of  the  gelatin  medium  are  that  it  is  perfectly 
transparent,  that  it  is  easily  melted  for  making  "  plates/'"  and  that 
many  bacteria  exhibit  in  it  special  characters  of  growth  by  which  they 
may  be  differentiated  from  others  which  resemble  them  in  form. 
The  principal  disadvantage  is  the  low  melting  point,  which  prevents 
us  from  making  use  of  this  medium  for  cultivating  bacteria  in  an  in- 
cubating oven  at  a  higher  temperature  than  about  22°  C.  for  ten-per- 
cent gelatin. 

This  disadvantage  is  overcome  by  using  agar-agar  instead  of 
gelatin.  This  is  prepared  in  Japan  and  other  Eastern  countries 
from  certain  species  of  gelatinous  algse.  It  comes  to  us  in  the  form 
of  bundles  of  dried  strips,  which  form  a  stiff  jelly  when  dissolved  in 
water  in  the  proportion  of  one  to  two  per  cent.  This  jelly  remains 
solid  at  a  temperature  of  40°  C.  and  above.  It  was  first  employed 
by  Hesse,  one  of  Koch's  collaborators  in  the  office  of  the  imperial 
board  of  health  of  Berlin.  Koch,  who  was  in  search  of  a  trans- 
parent jelly  which  would  stand  the  temperature  required  for  the  cul- 
tivation of  certain  pathogenic  bacteria  (37°  to  38°  C.),  quickly  recog- 
nized its  value  and  introduced  it  into  general  use. 

The  agar-agar  jelly  is  more  difficult  to  filter  than  the  gelatin 
medium,  and  some  skill  is  required  in  order  to  obtain  a  transparent 
solution.  It  will  bear  long  boiling  without  losing  its  quality  of 
forming  a  stiff  jelly.  From  ten  to  twenty  grammes  are  added  to  a 
litre  of  flesh  infusion,  or  we  may  make  a  peptonized  agar  in  accor- 
dance with  the  following  formula  which  is  given  by  Salomonson  : 
Add  to  one  litre  of  distilled  water  five  grammes  Liebig's  extract, 
thirty  grammes  peptone,  five  grammes  cane  sugar,  fifteen  grammes 
agar.  Cook  for  an  hour,  render  slightly  alkaline,  and  cool  to  below 
60°  C.  Clarify  and  cook  again  for  an  hour  or  more. 

Glycerin-cigar  is  made  by  adding  five  per  cent  of  glycerin  to 
the  peptonized  agar  made  by  the  above  formula  or  by  the  use  of  the 
flesh-peptone  infusion.  This  is  a  very  favorable  medium  for  the  cul- 
tivation of  the  tubercle  bacillus — first  used  by  Roux  and  Nocard. 


44  CULTURE    MEDIA. 

Agar-gelatin,  a  medium  which  has  recently  come  into  favor  and 
is  said  to  be  very  useful,  as  it  resembles  gelatin  in  transparency  and 
has  a  considerably  higher  melting  point  than  ten-per-cent  gelatin,  is 
made  by  adding  fifty  grammes  of  gelatin  and  7. 5  grammes  of  agar 
to  a  litre  of  flesh-peptone  solution.  Care  should  be  taken  not  to  cook 
this  longer  than  is  necessary. 

In  making  all  of  these  agar  culture  media  the  main  difficulties 
encountered  result  from  the  difficulty  of  dissolving  the  agar  and  the 
slowness  with  which  the  solution  passes  through  filtering  paper. 
These  difficulties  are  best  met  as  follows  :  Break  up  the  sticks  of  agar 
into  small  fragments  and  allow  them  to  soak  in  cold  water  for  twenty- 
four  hours.  Pour  off  the  water  and  add  the  flesh-peptone  solution. 
Boil  for  several  hours  until  the  agar  is  completely  dissolved.  Neu- 
tralize by  adding  gradually  a  solution  of  carbonate  of  soda  (or  render 
slightly  alkaline).  Filter. 

The  last  operation  is  the  most  troublesome,  and  various  plans 
have  been  proposed  to  avoid  the  tedious  filtration  through  filtering 
paper  'in  a  hot- water  filter.  A  method  which  gives  satisfactory  re- 
sults is  to  place  the  filter  containing  the  hot  agar  solution,  and  the 
flask  which  is  to  receive  the  filtrate,  in  a  steam  sterilizing  apparatus, 
where  it  is  left  in  an  atmosphere  of  streaming  steam  until  the  filtra- 
tion is  completed.  Or  the  solution  may  be  put  in  a  tall  jar  and  left 
in  the  steam  sterilizer  for  several  hours  until  it  is  clear  as  a  result  of 
sedimentation.  The  clear  solution  is  then  obtained  by  decantation. 
Or  by  conducting  the  operation  in  a  tall  cylindrical  vessel,  and  al- 
lowing sedimentation  to  occur  in  the  steam  sterilizer  and  the  agar 
subsequently  to  solidify  by  cooling,  the  cylinder  of  jelly  may  be  re- 
moved from  the  jar  and  the  part  containing  the  sediment  can  be  cut 
away.  The  transparent  portion  is  then  melted  again  and  distributed 
in  test  tubes  for  use. 

In  the  present  volume  we  frequently  refer  to  the  nutrient  medium 
made  by  adding  one  to  two  per  cent  of  agar-agar  to  the  standard 
flesh-peptone  solution  as  "  nutrient  agar"  or  simply  as  "agar." 

The  following  method  of  filtering  agar  has  (1890)  been  proposed 
by  Karlinsky.  It  is  a  modification  of  the  method  previously  de- 
scribed by  Jakobi  and  depends  upon  the  use  of  pressure. 

In  Fig.  18,  a  is  a  cylindrical  vessel  of  tin,  which  is  closed  above  by 
a  perforated  rubber  cork,  through  which  is  passed  a  glass  tube,  b. 
This  is  enclosed  in  a  larger  tin  cylinder,  c,  which  contains  water, 
which  may  be  kept  hot  by  placing  an  alcohol  lamp  under  the  pro- 
jecting arm  d.  The  central  cylinder  has  a  tube,  e,  passing  through 
the  bottom  of  the  hot- water  cylinder,  and  which  is  provided  with  a 
stopcock  for  drawing  off  the  filtered  solution.  Before  pouring  the 
hot  agar  solution  into  the  cylinder  a,  a  cotton  filter  about  ten  centi- 


CULTURE    MEDIA. 


45 


metres  thick  is  placed  at  the  bottom  of  this  cylinder  and  hot  water 
is  poured  upon  it  while  the  stopcock  of  the  outlet  tube  is  open.  This 
washes  out  the  cotton  and  prepares  the  filter  for  the  agar  solution. 
The  apparatus  is  supported  upon  a  tripod,  not  shown  in  the  figure. 
Filtration  is  said  to  occur  rapidly  when  the  air  in  the  central  cylinder 
is  compressed  by  means  of  the  hand  bellows  attached  to  the  tube  b. 


FIG.  18. 

Schultz*  Rapid  Method  of  Preparing  Sutrient  Agar- Agar. — 
Place  one  thousand  five  hundred  cubic  centimetres  of  water  in  an  en- 
amelled iron  pot;  add  eighteen  grammes  of  agar-agar,  broken  in  small 
pieces,  and  place  upon  a  gas  stove;  boil  for  half  an  hour;  add  while 
boiling  two  grammes  of  Liebig's  extract  of  beef ;  remove  from  fire  and 
cool  to  60°  C. ;  then  add  ten  grammes  of  dry  peptone,  five  grammes 
of  sodium  chloride,  and  the  contents  of  one  egg  beaten  up  in  a 
sufficient  quantity  of  water  to  supply  that  lost  by  evaporation;  neut- 
ralize the  mixture  by  the  addition  of  dilute  hydrochloric  acid;  boil 
again  for  five  or  ten  minutes;  filter  through  white  filter  paper.  If 
the  filtrate  is  not  entirely  clear  add  to  it  the  albumen  of  a  second 
egg  and  boil  until  this  is  coagulated  \  then  filter  again.  Always  mois- 
ten the  filter  ivitJi  water  before  filtering  solutions  containing 
gelatin  or  agar-agar.  When  the  process  is  completed  the  amount 
of  filtered  culture  medium  should  be  about  one  thousand  cubic  centi- 
metres. 


4t>  Ct'LTl'KE     MEDIA. 

According  to  Abbott  the  filtration  of  agar-agar  does  not  require 
the  use  of  a  hot-water  funnel  or  any  other  device  for  maintaining  the 
temperature  of  the  mass.  He  gives  the  following  directions  for  its 
preparation : 

"  Prepare  the  bouillon  in  the  usual  way.  Agar-agar  reacts  neutral  or 
very  slightly  alkaline,  so  that  the  bouillon  may  be  neutralized  before  the 
agar-agar  is  added.  Then  add  finely  chopped  or  powdered  agar-agar  in  the 
proportion  of  one  to  1.5  per  cent.  Place  the  mixture  in  a  porcelain-lined  iron 
vessel,  and  on  one  side  of  the  vessel  make  a  mark  at  the  height  at  which  the 
level  of  the  fluid  stands.  If  a  litre  of  medium  is  being  made,  add  about  two 
hundred  and  fifty  to  three  hundred  cubic  centimetres  more  water,  and  allow 
the  mass  to  boil  slowly,  occasionally  stirring,  over  a  free  flame,  from  one  and 
a  half  to  two  hours;  or  until  the  excess  of  water — i.e.,  the  two  hundred  and 
fifty  or  three  hundred  cubic  centimetres  that  were  added — has  evaporated. 
Care  must  be  taken  that  the  liquid  does  not  boil  over  the  sides  of  the  vessel. 
From  time  to  time  observe  if  the  fluid  has  fallen  below  its  original  level  ;  if 
it  has,  add  water  until  its  volume  of  one  litre  is  restored.  At  the  end  of  the 
time  given  remove  the  tlameand  place  the  vessel  containing  the  mixture  in  a 
large  dish  of  cold  water  ;  stir  the  agar-agar  continuously  until  it  has  cooled 
to  about  68  to  70  C.,  and  then  add  the  white  of  one  egg  which  has  been 
beaten  up  in  about  fifty  cubic  centimetres  of  water;  or  the  ordinary  dried  al- 
bumeiiof  commerce  maybe  dissolved  in  cold  water  in  the  proportion  of  about 
ten  per  cent  and  used — the  results  are  equally  as  good  as  when  eggs  are  em- 
ployed. Mix  this  carefully  throughout  the  agar-agar  and  allow  the  mass  to 
boil  slowly  for  about  another  half-hour,  observing  a  11  the  while  the  level  of  the 
fluid,  which  should  not  fall  below  the  litre  mark.  It  is  necessary  to  reduce  the 
temperature  of  the  mass  to  the  point  given,  68"  to  70  C.  ;  otherwise  the  co- 
agulation of  the  albumen  will  occur  suddenly  in  lumps  and  masses  as  soon 
as  it  is  added,  and  its  clearing  action  will  not  be  uniform.  The  process  of 
clarification  with  the  egg  is  purely  mechanical ;  the  fine  particles,  which 
would  otherwise  pass  through  the  pores  of  the  filter,  being  taken  up  by  the 
albumen  as  it  coagulates  and  being  retained  in  the  coagula.  At  the  end  of 
one- half  hour  the  boiling  mass  may  be  easily  and  quickly  filtered  through  a 
heavy-folded  paper  filter  at  the  room  temperature." 

For  special  purposes  various  substances  are  added  to  the  above- 
described  solid  and  liquid  media.  A  favorable  addition  for  the 
growth  of  a  considerable  number  of  bacteria  is  from  one  to  three  per 
cant  of  glucose.  The  phosphorescent  bacteria  grow  best  in  a  minlmm 
containing  two  to  three  per  cent  of  sodium  chloride.  The  addition 
of  three  to  four  per  cent  of  itotitxxinin-  n  it  rate  is  made  in  conducting 
experiments  designed  to  test  the  reducing  power  of  certain  bacteria, 
by  which  this  salt  is  decomposed  with  the  production  of  nitrites. 
Acids  are  also  added  in  various  proportion  to  test  the  ability  of 
bacteria  under  investigation  to  grow  in  an  acid  medium.  From 
1 :  2,000  to  1 :  500  of  hydrochloric  acid  may  be  used  for  this  purpose. 
The  addition  of  litmus  to  milk  or  other  culture  media  is  fre- 
quently resorted  to  for  the  purpose  of  ascertaining  whether  acids  or 
alkalies  are  developed  during  the  growth  of  bacteria  under  investi- 
gation. The  addition  of  an  if  in  c  culorx  which  are  variously  changed 
by  the  products  of  growth  of  certain  spiv'u's  has  also  been  resorttnl 
to  in  the  differentiation  of  species.  Various  disinfecting  agents,  such 
1  Abbott's  "Principles  of  Bacteriology  "  Fifth  edition,  pp.  100  and  101. 


CULTURE    MEDIA.  47 

as  carbolic  acid,  etc.,  have  also  been  used  for  the  same  purpose,  and 
it  has  been  shown  by  experiment  that  some  bacteria  will  grow  in  a 
medium  containing  such  agents  in  a  proportion  which  would  entirely 
restrain  the  development  of  others. 

The  soluble  silicates  which  form  a  jelly-like  mass  have  been 
proposed  as  a  culture  medium  for  certain  bacteria  which  do  not  grow 
in  the  usual  media.  Kuhne  (1890),  Winogradsky  (1891),  and  Sles- 
kin  (181)1)  have  made  experiments  which  indicate  that  this  medium 
has  considerable  value. 

Winogradsky  uses  in  the  preparation  of  his  silicate  jelly  the 
following  salts  : 

Ammonium  sulphate,  ....  0.4  gramme. 

Magnesium  sulphate,     ....  0.05 

Potassium  phosphate,          ....  0.1 

Calcium  chloridf,          ....  a  trace. 

Sodium   carbonate,  .  .  .  0.6  to  0.9  gramme. 

Distilled  water,  .  .  .  .       .  100  grammes. 

To  this  he  adds  a  solution  of  silicic  acid.  According  to  Kiihne,  a 
solution  containing  3.4  per  cent  of  silicic  acid  and  having  a  specific 
gravity  of  1.02  may  be  preserved  in  a  liquid  condition.  To  this  the 
salts  are  added  in  greater  or  less  amount,  according  to  the  consis- 
tence desired. 

Sleskiii  states  that  a  suitable  jelly  is  formed  by  the  addition  of 
1.15  to  1.45  per  cent  of  the  salts,  and  recommends  that  concentrated, 
sterilized  solutions  be  added  to  the  acid.  He  dissolves  separately,  in 
as  little  water  as  possible,  the  sulphates,  the  potassium  phosphate 
and  sodium  carbonate,  and  the  calcium  chloride. 

The  use  of  a  culture  medium  containing  an  extract  from  the  je- 
quirity  seeds  has  been  recommended  by  Kaufmann  (1891),  who  has 
found,  by  experimenting  upon  various  bacteria,  that  such  a  medium 
is  useful  in  differentiating  species. 

TliejeqHt'rity  solution,  which  may  be  used  as  a  liquid  medium 
or  may  be  employed  in  the  preparation  of  nutrient  gelatin  or  agar,  is 
prepared  as  follows  :  Ten  grammes  of  jequirity  seeds  are  bruised  in 
a  mortar  and  the  shells  removed  ;  they  are  then  placed  in  one  hun- 
dred cubic  centimetres  of  water  and  cooked  for  two  hours  in  the  steam 
sterilizer ;  after  allowing  the  infusion  to  cool  it  is  filtered.  The  fil- 
tered liquid  has  a  pale-yellow  color  and  a  neutral  or  slightly  alkaline 
reaction.  Certain  bacteria  grow  in  this  solution  without  producing 
any  change  in  its  color ;  others,  which  produce  an  acid  reaction, 
cause  it  to  be  decolorized  ;  others,  which  produce  an  alkaline  reac- 
tion of  the  medium,  change  the  color  to  green. 

Lactose  Litmus- Agar. — Trfts  medium  is  useful  for  the  detection 
of  the  typhoid  bacillus  in  mixed  cultures,  e.g.,  in  faeces.  It  is  made 
by  adding  to  nutrient  agar-agar,  having  a  slightly  alkaline  reaction, 


48  CULTURE    MEDIA. 

two  or  three  per  cent  of  lactose  and  enough  tincture  of  litmus  to  give 
the  culture  medium  a  pale  blue  color.  Colonies  of  bacteria  growing 
in  this  medium  which  cause  a  fermentation  of  the  lactose,  with 
formation  of  acid,  have  a  pale  pink  color,  extending  to  the  surround- 
ing medium.  Colonies  which  do  not  give  rise  to  acid  production 
are  pale  blue.  Thus,  colonies  of  the  colon  bacillus  would  be  red  and 
colonies  of  the  typhoid  bacillus  blue. 

Blood-serum  Mixture  of  Loffler. — This  consists  of  three  parts 
blood  serum  and  one  part  of  neutral  meat  infusion,  containing  one  per 
cent  of  glucose.  It  is  sterilized  and  solidified  as  directed  for  blood 
serum,  but  a  higher  temperature  is  required  for  coagulation  of  the 
mixture  than  for  plain  blood  serum. 

Cooked  Potato. — Schroter  first  used  cooked  potato  as  a  culture 
medium  for  certain  chromogenic  bacteria  (1872),  and  Koch  subse- 
quently called  attention  to  the  great  value  of  potato  cultures  for 
differentiating  species.  His  plan  of  preparing  potatoes  is  as  follows : 
Sound  potatoes  are  chosen  in  which  the  epidermis  is  intact.  These 
are  thoroughly  washed  and  scrubbed  with  a  brush  to  remove  all 
dirt.  The  "  eyes"  and  any  bruised  or  discolored  spots  are  removed 
with  a  sharp-pointed  knife.  They  are  again  thoroughly  washed  in 
water,  and  are  then  placed  for  an  hour  in  a  bath  containing 
mercuric  chloride  in  the  proportion  of  1 :  500,  to  thoroughly  disinfect 
the  surface.  They  are  then  placed  in  a  steam  sterilizer  for  about 
three-quarters  of  an  hour,  and  after  an  interval  of  twenty-four  hours 
are  again  steamed  for  fifteen  minutes.  It  is  well  to  wrap  each 
potato  in  tissue  paper  before  placing  it  in  the  bichloride  bath,  and  to 
leave  it  in  this  protecting  envelope  until  it  is  placed  in  the  glass  dish 
in  which  it  is  preserved  from  contamination  by  atmospheric  germs 
after  being  inoculated  with  some  particular  microorganism.  Just 
before  such  inoculation  the  potato  is  cut  in  halves  with  a  sterilized 
(by  heat)  table  knife.  The  bacteria  to  be  cultivated  are  placed  upon 
the  cut  surface  and  the  potato  is  preserved  in  a  glass  dish  (Fig.  20). 

A  more  convenient  method,  and  one  which  secures  the  potato  more 
effectually  from  atmospheric  organisms,  is  to  cut  a  cylinder,  about 
an  inch  in  diameter,  from  a  sound  potato,  by  means  of  a  tin  instru- 
ment resembling  a  cork  borer  or  apple  corer.  This  cylinder  is  cut 
obliquely  into  two  pieces  having  the  form  shown  in  Fig.  22,  and 
each,  piece  is  placed  in  a  large  test  tube  having  a  cotton  air  filter,  in 
which  it  is  sterilized.  This  method,  first  employed  by  Boltoii,  has 
been  slightly  modified  by  Roux,  who  recommends  that  a  receptacle 
for  catching  the  water  which  separates  during  the  sterilizing  process 
be  formed  by  making  a  constriction  around  the  test  tube  an  inch 
above  its  lower  extremity.  This  is  done  by  the  use  of  a  blowpipe. 


CULTURE    MEDIA. 


49 


The  cylinder  of  potato  rests  upon  the  constricted  portion  of  the  tube, 
as  shown  in  Fig.  21. 

Sometimes  a  potato  paste  is  employed.  The  potatoes  are  boiled 
for  an  hour  and  the  skins  removed,  after  which  they  are  mashed 
with  a  little  sterilized  water,  placed  in  suitable  plates,  and  sterilized 
by  exposure  for  half  an  hour  on  three  successive  days  in  the  steam 
sterilizer.  Bread  paste  may  be  made  in  the  same  way,  and  is  a  very 
favorable  medium  for  the  growth  of  certain  bacteria  and  also  for  the 
common  moulds. 


FIG.  20. 


FIG.  21. 


FIG.  22. 


Neutralization  of  Culture  Media.  —  For  ordinary  purposes 
neutralization  of  acid  culture  media  is  accomplished  by  the  use  of  a 
saturated  solution  of  sodium  carbonate,  the  reaction  being  tested  with 
strips  of  blue  and  red  litmus  paper.  But  for  certain  investigations 
it  is  essential  that  a  more  sensitive  and  reliable  indicator  should  be 
used,  and  that  an  exact  method  of  titration  be  employed.  Schultz 
(1891)  recommends  the  use  of  phenolphthalein  as  an  indicator  and 
titration  with  a  solution  of  caustic  soda  (four-per-cent  stock  solution, 
to  be  diluted  to  0.4  per  cent  for  use).  One  drop  of  phenolphthalein 
solution,  containing  one  gramme  to  three  hundred  cubic  centimetres 


50  CULTURE    MEDIA. 

of  alcohol,  should  be  added  to  one  cubic  centimetre  of  bouillon.  The 
beginning  of  an  alkaline  reaction  is  indicated  by  the  appearance  of  a 
faint  rose  color.  Fuller  (1895),  who  has  made  a  careful  investiga- 
tion of  this  subject,  recommends  a  modification  of  the  method  of 
Schultz.  He  gives  the  following  directions  in  his  paper  published 
in  the  Journal  of  the  American  Public  Health  Association  (Vol. 
XX.,  p.  386): 

This  indicator  is  prepared  by  dissolving-  five  grammes  of  commercial 
phenolphthalein in  one  litre  of  fifty-per-cent  alcohol.  It  is  not  feasible  to  use 
this  indicator  011  strips  of  paper  as  the  alcohol  quickly  evaporates,  leaving  the 
powder.  The  colorless  liquid,  however,  may  be  added  in  small  quantities  to 
solutions  of  an  acid  or  neutral  nature  without  any  change  of  color,  but  alka- 
lies quickly  change  it  to  a  purple  red.  This  change  from  110  color  to  one  of 
purple  red  makes  the  indicator  a  very  satisfactory  one,  owing  to  the  ease 
with  which  the  eye  detects  the  so-called  end- point. 

For  the  determination  of  the  degree  of  reaction  of  nutrient  media  it  is 
the  custom  to  put  five  cubic  centimetres  (practically  five  drachms)  of  the  solu- 
tion into  a  six-inch  porcelain  evaporating  dish,  together  with  forty-five  cubic 
centimetres  of  distilled  water.  This  liquid  is  boiled  for  three  minutes,  after 
which  is  added  one  cubic  centimetre  of  the  phenolphthalein  solution.  While 
the  solution  is  still  hot  it  is  quickly  titrated  against  a  twentieth  normal  solu- 
tion of  caustic  alkali. 

As  already  mentioned,  Dahmen  stated  that  this  indicator  is  useless,  ow- 
ing to  the  presence  of  carbonates,  and  of  ammonia  and  its  salts.  On  gen- 
eral grounds  the  point  that  this  indicator  is  inaccurate  under  such  conditions 
is  well  taken,  but  so  far  as  its  application  to  nutrient  media  is  concerned  it 
has  been  found  that  by  proper  precautions  these  objections  may  be  over- 
come. 

With  regard  to  the  amount  of  free  and  combined  ammonia  present  in 
ordinary  nutrient  media  at  the  times  when  their  reaction  is  determined,  it 
has  been  found  that  it  does  not  exceed  0.003  per  cent.  Experiments  show 
that  this  quantity  is  less  than  one- tenth  of  that  necessary  to  interfere  with 
the  accuracy  of  the  method.  It  may  be  added  that  the  reason  why  no  am- 
monia is  produced  by  the  addition  of  alkali  to  the  nitrogenous  bodies  is  that 
at  no  time  during  the  preparation  is  there  an  appreciable  excess  of  free  alkali 
present. 

The  chief  point  by  which  the  presence  of  carbon  dioxide  is  obviated  is  by 
the  use  of  caustic  soda  instead  of  sodium  carbonate  for  neutralization,  as  re- 
ferred to  beyond.  It  has  been  learned  by  actual  experiment  that  the  carbon 
dioxide  is  practically  all  removed  by  heat  during  the  preparation  of  the 
media  and  at  the  time  of  boiling  just  prior  to  the  titration.  In  order  to  pre- 
vent atmospheric  absorption  of  this  gas  the  titration  should  be  made  quickly 
and  in  a  hot  solution. 

The  remaining  precaution  concerns  the  solution  against  which  the  nu- 
trient media  are  titrated.  All  of  the  usual  media  react  acid  to  phenolphtha- 
lein ;  hence  the  solution  in  question  must  be  alkaline.  Caustic  soda  serves 
the  purpose  well,  and  the  strength  may  be  conveniently  one-twentieth  normal, 
equal  to  two  grammes  per  litre.  For  the  sake  of  prevention  of  interference 
from  carbon  dioxide  in  the  standard  solution  as  it  meets  the  indicator,  it  is 
well  to  add  a  small  quantity  of  calcium  hydrate  in  order  to  precipitate  this 
gas  as  calcium  carbonate  and  allow  it  to  settle  out  in  the  reagent  bottle. 
After  this  solution  has  been  accurately  prepared,  care  is  necessary  in  order 
to  keep  it  of  uniform  strength  and  free  from  carbon  dioxide.  This  is  best 
done  by  placing  the  stock  solution  bottle  on  a  shelf  from  which  the  liquid 
may  be  delivered  into  the  burette  by  means  of  a  siphon  that  is  connected 
tightly  with  the  top  of  the  burette.  In  the  tightly  fitting  stopper  of  the  bottle 


CULTURE    MEDIA.  51 

are  three  perforations :  one  through  which  the  siphon  passes,  and  another 
for  a  U  tube  filled  with  concentrated  caustic  soda  in  order  to  absorb  the 
carbon  dioxide  from  all  the  air  which  enters  the  bottle.  The  third  perfora- 
tion is  for  a  by-pass  which  connects  with  the  siphon  just  above  the  top  of  the 
burette  and  below  the  cock  by  which  the  flow  from  the  bottle  is  regulated. 
The  object  of  this  is  to  provide  for  the  entrance  into  the  burette,  as  the  solu- 
tion is  allowed  to  run  from  it,  of  air  that  has  passed  through  the  U  tube  and 
has  had  its  carbon  dioxide  removed. 

When  the  manipulation  is  carried  out  uniformly  in  the  manner  thus  de- 
scribed, and  with  the  constant  employment  of  an  end-point  which  has  the 
same  intensity  of  color,  very  satisfactory  and  closely  agreeing  results  may 
be  obtained  by  this  method. 


VI. 
STERILIZATION  OF   CULTURE   MEDIA. 

A  MOST  important  part  of  bacteriological  technology  consists  in 
the  sterilization  of  the  various  culture  media  employed.  A  sterile 
medium  is  essential  for  maintaining  a  pure  culture,  and  we  can  only 
obtain  an  exact  knowledge  of  the  biological  characters  of  a  species 
by  studying  its  growth  in  various  media,  its  physiological  reactions, 
its  pathogenic  power,  etc.,  independently  of  all  other  microorgan- 
isms— i.  e. ,  in  pure  cultures. 

We  may  sterilize  a  culture  medium  either  by  heat  or  by  filtration 
through  a  substance  which  does  not  permit  bacteria  to  pass.  The 
last-mentioned  method  is  useful  for  certain  special  purposes  ;  but,  in 
general,  sterilization  of  culture  media,  and  of  the  vessels  in  which 
they  are  preserved,  is  effected  by  heat. 

The  scientific  use  of  heat  as  an  agent  for  sterilizing  our  culture 
media  depends  upon  a  knowledge  of  the  thermal  death-point  of  the 
various  microorganisms  which  are  liable  to  be  present  in  them,  and 
upon  various  facts  relating  to  the  manner  in  which  heat  is  applied. 
All  this  has  been  determined  by  experiment,  and  before  giving 
practical  directions  for  sterilization  it  will  be  well  to  consider  the 
experimental  data  upon  which  our  methods  are  based. 

As  a  rule,  bacteria  which  do  not  form  spores  are  killed  at  a  com- 
paratively low  temperature.  Thus,  in  a  series  of  experiments  made 
by  the  writer  upon  the  thermal  death-point  of  various  pathogenic 
organisms,  the  pus  cocci  were  found  to  be  the  most  resistant,  and  all 
of  these  were  killed  by  exposure  for  ten  minutes  to  a  temperature 
of  62°  C.  (143.6°  F.).  There  are  several  species  of  bacteria  known, 
however,  which  not  only  are  not  killed  by  this  temperature,  but  are 
able  to  grow  and  multiply  at  a  temperature  of  65°  to  70°  C.  (Miquel, 
Van  Tieghem,  Globig).  But  it  is  safe  to  say  that  exposure  to  a 
boiling  temperature  for  a  minute  or  two  will  infallibly  destroy  all 
microorganisms  in  the  absence  of  spores,  when  they  are  in  a  moist 
condition  or  moist  heat  is  used — i.e.,  when  they  are  directly  ex- 
posed to  the  action  of  boiling  water  or  of  steam.  The  power  of  dry 
heat  to  destroy  microorganisms  in  a  desiccated  condition  is  a  differ- 
ent matter  and  will  require  special  consideration. 


STERILIZATION    OF    CULTURE    MEDIA.  53 

The  spores  of  bacilli  have  a  much  greater  resisting  power,  and 
the  vitality  of  some  of  these  reproductive  bodies,  from  known  spe- 
cies, is  not  destroyed  by  a  boiling  temperature  maintained  for  sev- 
eral hours.  Thus  Globig  found  that  the  spores  of  a  certain  bacillus 
from  the  soil — his  "  red  potato  bacillus  "  —required  six  hours'  exposure 
to  streaming  steam  in  order  to  destroy  it.  Steam  under  pressure,  at 
a  temperature  of  115°  C.,  killed  it  in  half  an  hour  ;  at  125°  C.  in  five 
minutes.  This  extreme  resisting  power  is  exceptional,  however, 
and  many  spores  are  destroyed  in  a  few  minutes  by  the  boiling  tem- 
perature of  water. 

In  practice  we  assume  that  some  of  the  more  resistant  spores, 
which  are  frequently  present  in  the  atmosphere,  may  have  fallen 
into  our  culture  material,  and  to  insure  its  sterilization  we  subject  it 
to  a  temperature  which  can  be  depended  upon  to  destroy  these  ;  or 
we  resort  to  the  method  of  discontinuous  heating.  This  method 
was  first  employed  by  Tyndall  (1877),  and  is  now  in  general  use  in 
the  bacteriological  laboratories  of  Germany,  having  been  adopted  by 
Koch  and  his  pupils ;  while  in  France  a  single  sterilization  by  means 
of  steam  under  pressure,  securing  a  higher  temperature,  is  still  the 
favorite  method  with  many. 

In  the  method  by  discontinuous  heating  we  subject  the  culture 
material  for  a  short  time  to  the  temperature  of  boiling  water,  thus 
destroying  all  bacteria  in  the  vegetative  stage.  After  an  interval, 
usually  of  twenty-four  hours,  we  repeat  the  operation  for  the  pur- 
pose of  destroying  those  which  in  the  meantime  have  developed 
from  spores  which  may  have  been  present.  Again  the  material  is 
put  aside,  and  after  twenty-four  hours  it  is  again  heated  to  the 
boiling  point.  This  is  usually  repeated  from  three  to  five  times. 
The  object  in  view  is  to  kill  the  growing  bacteria  which  are  de- 
veloped from  spores  which  were  present ;  and,  as  a  matter  of  expe- 
rience, we  find  that  this  method  of  sterilization  is  more  reliable  than 
a  single  prolonged  boiling,  unless  this  be  effected  at  a  higher  tem- 
perature than  that  of  boiling  water  at  the  ordinary  pressure  of  the 
atmosphere.  Discontinuous  heating  is  especially  useful  for  the  sterili- 
zation of  liquids  which  would  be  injured  by  prolonged  boiling — as  is 
the  case  with  solutions  of  gelatin — or  which  are  coagulated  by  the 
boiling  temperature.  By  means  of  a  water  bath,  the  temperature 
of  which  is  regulated  automatically,  we  may  conduct  the  operation 
at  any  desired  degree.  Thus  in  sterilizing  blood  serum  we  use  a 
temperature  a  little  below  that  at  which  coagulation  occurs  (about 
70°  C.). 

Test  tubes,  flasks,  and  apparatus  of  various  kinds  are  commonly 
sterilized  by  dry  heat  in  &  hot-air  oven.  This  is  usually  made  of 
sheet  iron,  with  double  walls,  and  shelves  for  supporting  the  articles 


54  STERILIZATION   OF   CULTURE    MEDIA. 

to  be  sterilized.  The  form  shown  in  Fig.  23  is  commonly  used  in 
bacteriological  laboratories. 

It  must  be  remembered  that  a  much  higher  temperature  is  re- 
quired for  the  destruction  of  microorganisms  when  dry  heat  is  em- 
ployed than  is  the  case  with  moist  heat.  The  experiments  of  Koch 
and  Wolffhiigel  (1881)  show  that  a  temperature  of  120°  to  128°  C. 
(348°  to  262°  F.)  is  required  to  destroy  the  spores  of  mould  fungi,  and 
micrococci  or  bacilli  in  the  absence  of  spores.  For  the  spores  of  ba- 
cilli a  temperature  of  140°  C.  (284°  F.),  maintained  for  three  hours, 
was  required. 

In  practice  we  usually  maintain  a  temperature  of  about  150°  C. 


FIG.  23. 

(302°  F.)  for  an  hour  or  more;  and  it  is  customary  to  sterilize  all 
test  tubes  and  flasks,  which  are  to  be  used  as  receptacles  for  culture 
media,  in  the  hot-air  sterilizer.  This  procedure  could  no  doubt,  how- 
ever, be  dispensed  with  in  many  cases  and  reliance  be  placed  upon 
the  sterilization  of  the  flask,  together  with  its  contents,  in  the  steam 
sterilizer,  especially  with  such  culture  media  as  are  not  injured  by 
long  exposure  to  a  boiling  temperature — e.  g. ,  bouillon  and  agar-agar. 
When  we  propose  to  cultivate  aerobic  bacteria,  or  such  as  require 
oxygen  for.  their  development,  a  cotton  air  filter  is  placed  in  the 
mouth  of  each  test  tube  and  flask  before  it  is  sterilized  in  the  hot-air 
oven.  This  is  a  loose  plug  of  cotton,  pushed  into  the  neck  of  the 
flask  for  an  inch  or  more,  and  projecting  from  its  mouth  for  a  short 
distance.  These  cotton  filters  should  fill  the  tube  completely  and 


STERILIZATION   OF   CULTURE   MEDIA. 


55 


uniformly,  but  should  not  be  packed  so  closely  that  there  is  difficulty 
is  removing  them. 

Steam  Sterilizers. — Steam  at  the  ordinary  pressure  of  the  atmo- 
sphere has  the  same  temperature  as  boiling  water,  and  in  practice  is 
preferable  to  a  water  bath  for  several  reasons.  The  form  of  steam 
sterilizer  adopted  by  Koch,  after  extensive  experiments  made  in  col- 
laboration with  Loffler  and  Gaff ky,  is  now  generally  used  in  bacte- 
riological laboratories.  This  is  shown  in  Fig.  24.  It  consists  of  a 
cylindrical  vessel  of  zinc  which  is  covered  with  a  jacket  of  felt. 
The  cover,  also  covered  with  non-conducting  material,  has  an  aper- 
ture at  the  top  for  the  escape  of  steam.  A  glass  tube,  which  is  in 
communication  with  the  interior  of  the  vessel,  serves  to  show  the 


FIG.  24. 


FIG.  25. 


height  of  the  water  when  the  apparatus  is  in  use.  The  bottom  of 
the  cylindrical  vessel  should  be  of  copper.  A  Bunsen  burner  having 
three  jets  will  commonly  be  required  to  keep  the  water  in  ebullition 
and  the  upper  part  of  the  steam  sterilizer  rilled  with  ' '  live  steam," 
which  should  escape  freely  from  the  aperture  in  the  cover  to  insure 
a  temperature  of  100°  C.  in  the  steam  chamber.  A  perforated  zinc 
or  copper  shelf  in  the  interior  of  the  cylinder  serves  to  support  the 
flasks,  etc.,  which  are  to  be  sterilized.  Usually  they  are  lowered 
into  the  cylinder  in  a  light  wire  basket,  or  tin  pail  with  perforated 
bottom,  of  proper  diameter  to  slip  easily  into  the  sterilizer. 

Fig.  25  is  a  sectional  view  of  this  sterilizer. 

The  steam  sterilizer  shown  in  Fig.  26  '  is  an  American  invention, 

1  The  Arnold  steam  sterilizer,  manufactured  at  Rochester,  X.  Y. 


56 


STERILIZATION   OF   CULTURE   MEDIA. 


which  answers  the  purpose  admirably,  and  which  has  the  advantage 
of  getting  up  steam  very  quickly  and  also  of  using  comparatively 
little  gas. 

The  use  of  steam  under  pressure,  by  which  higher  temperatures 
are  obtained,  requires  a  more  expensive  apparatus,  made  on  the 
principle  of  Papin's  digester.  The  form  manufactured  by  Muncke 
is  one  of  the  best.  This  is  shown  in  Fig.  27.  It  is  provided  with  a 
pressure  gauge  and  a  safety  valve.  A  single  sterilization  in  this  ap- 
paratus, at  a  temperature  of  115°  C.,  for  half  an  hour,  will  usually 


M 


FIG.  26. 


FIG.  27. 


suffice,  and  for  liquid  culture  media  or  for  agar-agar  this  method  is 
entirely  satisfactory  ;  but  a  gelatin  medium  which  is  exposed  to  this 
temperature  loses  its  property  of  forming  a  jelly  at  20°  to  22°  C.,  and 
consequently  its  value  as  a  solid  culture  medium.  In  practice  the 
simpler  form  of  apparatus  in  which  streaming  steam  is  used  will  be 
found  ^to  answer  every  requirement.  To  insure  sterilization  with 
this  it  is  customary  to  resort  to  discontinuous  heating,  as  heretofore 
described.  The  standard  flesh-peptone-gelatin  medium  should,  as 
a  rule,  be  subjected  to  a  temperature  of  100°  C.  for  ten  minutes,  at 
intervals  of  twenty-four  hours,  four  days  in  succession.  Bouillon, 
flesh  infusions,  and  agar-agar  jelly  may  be  steamed  for  an  hour  at  a 
time  two  or  three  days  in  succession. 


STERILIZATION   OF   CULTURE   MEDIA.  57 

It  is  always  advisable  to  test  the  sterilization  of  culture  material 
before  making  use  of  it.  This  is  done  by  placing  it  for  a  few  days 
in  an  incubating  oven  at  30°  to  35°  0.  If  a  considerable  quantity  of 
material  in  test  tubes  has  been  prepared  at  one  time,  it  will  be  suffi- 
cient to  put  a  few  tubes  in  the  incubating  oven  to  test  sterilization. 

Failure  to  make  this  test  often  leads  to  serious  complications  in 
experimental  investigations.  A  laboratory  sometimes  becomes  in- 
fected with  resistant  spores,  which  are  not  all  destroyed  by  the  usual 
methods  of  sterilization,  and  these  may  not  develop  until  some  time 
has  elapsed  after  the  supposed  sterilization. 

Sterilization  of  Blood  Serum. — Blood  serum  which  has  been 
collected  in  test  tubes  or  small  flasks,  as  heretofore  directed,  is 


FIG.  28. 

sterilized  in  a  water  bath  at  60°  C.  (140°  F.)  by  the  method  of  dis- 
continuous heating.  It  is  usually  left  in  the  hot- water  bath  for 
about  an  hour,  and  this  is  repeated,  at  intervals  of  twenty-four  hours, 
for  five  to  seven  days.  This  rather  tedious  process  may  be  avoided 
by  collecting  the  serum  in  the  first  instance  with  proper  precautions 
to  prevent  it  from  becoming  contaminated  with  atmospheric  organ- 
isms. A  special  apparatus  was  devised  by  Koch  for  sterilizing  blood 
serum,  but  an  improvised  hot- water  bath  which  is  regulated  to  a 
temperature  of  60°  C.  by  an  automatic  thermo-regulator  will  answer 
the  purpose.  After  being  sterilized,  the  serum  is  solidified  by  careful 
exposure  to  a  temperature  of  about  68°  C.,  which  causes  it  to  CO' 
agulate,  forming  a  transparent,  jelly-like  mass.  When  coagulated 
at  a  higher  temperature  it  becomes  opaque.  The  time  required  for 
this  operation  varies  from  half  an  hour  to  an  hour,  and  it  is  best  to 
remove  the  tubes  from  the  receptacle  in  which  they  are  exposed  to 


58  STERILIZATION    OF    CULTURE    MEDIA. 

heat  as  soon  as  the  serum  is  solidified.  Koch's  apparatus  for  coagu- 
lating blood  serum  is  shown  in  Fig.  28.  It  is  customary  to  place  the 
test  tubes  in  an  oblique  position,  so  that  a  large  surface  may  be  ex- 
posed upon  which  to  cultivate  the  tubercle  bacillus  or  whatever 
microorganism  may  be  under  investigation.  A  form  of  apparatus 
designed  for  both  sterilizing  and  coagulating  blood  serum  is  shown 
in  Fig.  29.  It  is  manufactured  by  Miincke  in  accordance  with  the 
directions  of  Hueppe,  and  special  precautions  have  been  taken  to  se- 
cure a  uniform  temperature  in  all  parts  of  the  air  chamber.  We 


FIG.  2ft. 

may  remark  that  since  it  has  been  shown  by  Roux  and  Nocard  that 
the  tubercle  bacillus  grows  very  well  in  agar-agar  jelly  to  which 
five  per  cent  of  glycerin  has  been  added,  blood  serum  is  not  so 
largely  used  as  a  culture  medium  in  bacteriological  laboratories. 

Sterilization  by  Filtration.-*-This  method  is  especially  useful 
for  separating  the  soluble  substances  contained  in  a  liquid  culture  of 
bacteria  from  the  living  cells.  It  has  been  demonstrated  that  several 
of  the  most  important  pathogenic  bacteria  produce  toxic  substances 
during  their  growth  which  may  cause  the  death  of  susceptible  ani- 
mals independently  of  the  living  bacteria ;  and  this  demonstration 


STERILIZATION   OF   CULTURE   MEDIA. 


59 


has  been  made  either  by  sterilizing  a  pure  culture  by  means  of  heat, 
or  by  separating  the  bacteria  from  the  culture  "liquid  by  nitration. 
Some  of  these  toxic  products  of  bacterial  growth  are  destroyed  by  a 
comparatively  low  temperature  ;  the  method  of  sterilization  by  ni- 
tration is  therefore  very  important  in  researches  relating  to  the 
composition  and  pathogenic  power  of  these  soluble  products.  Pas- 
teur, in  his  earlier  experiments,  used  plaster  of  Paris  as  a  filter,  and 


Fig.  30. 


subsequently  resorted  to  the  use  of  unglazed  porcelain,  through 
which  a  liquid  may  be  forced  by  pressure,  but  which  does  not  per- 
mit of  the  passage  of  suspended  particles,  however  small. 

As  the  porcelain  filter  is  the  most  reliable  and  convenient  for 
accomplishing  the  object  in  view,  we  shall  not  describe  other  methods 
of  filtration  which  have  been  proposed  and  successfully  used.  The 
porcelain  used  is  a  very  fine  paste,  manufactured  at  Sevres,  which  is 
moulded  into  cylinders  (bougies)  of  the  form  proposed  by  Chamber- 
land  and  baked  at  a  high  temperature. 


60 


STERILIZATION   OF   CULTURE   MEDIA. 


In  Fig.  30  the  Pasteur-Chamberland  filter  is  shown  as  arranged 
for  the  filtration  of  -water.  A  is  the  hollow  porcelain  cylinder,  which 
is  enclosed  in  a  metal  case,  D.  The  metal  case  is  tightly  clamped 
against  a  projecting  shoulder  at  the  lower  part  of  the  porcelain  filter, 
a  ring  of  rubber  being  interposed  to  secure  a  tight  joint.  When 
water  under  pressure  is  admitted  to  the  space  E,  between  the  cylin- 
der of  porcelain  and  the  metal  case,  it  slowly  filters  through,  and, 
running  down  the  inner  wall  of  the  filter,  escapes  at  B  into  a  recep- 
tacle placed  to  receive  it.  If  we  fill  the  space  E  with  a  liquid  cul- 
ture of  bacteria  and  apply  sufficient  pressure  (one  or  two  atmo- 
spheres), a  clear  filtrate  is  obtained  which  is  entirely  sterile  if  the 
porcelain  filter  is  sound  and  made  of  proper  material.  After  the 


FIG.  31. 

filter  has  been  in  use  for  some  time,  however,  it  may  permit  the  pas- 
sage "of  bacteria,  and  it  will  be  necessary  to  subject  it  to  a  high  tem- 
perature for  the  purpose  of  destroying  all  organic  matter  contained 
in  the  porous  porcelain. 

We  may  use  the  Chamberland  filter  without  a  metal  case  by  im- 
mersing it  in  a  cylindrical  glass  vessel  containing  the  liquid  to  be  fil- 
tered, as  shown  in  Fig.  31.  The  porcelain  cylinder  is  connected  with 
an  aspirator  bottle,  «,  and  a  small  Eiienmeyer  flask,  b,  is  interposed 
to  catch  the  filtrate  when  it  overflows  from  the  interior  of  the  filter. 
Of  course  all  the  necessary  precautions  must  be  taken  with  reference 
to  the  sterilization  of  the  interior  of  the  bougie,  of  the  flask  b,  and  of 
the  rubber  tube  connecting  the  two. 

Another  arrangement  of  the  Pasteur-Chamberland  filter  for  labora- 
tory purposes  is  shown  in  Fig.  32.  In  this  form  of  apparatus  a 


STERILIZATION   OF   CULTURE   MEDIA. 


61 


receptacle,  R,  is  provided  for  the  liquid  to  be  filtered,  and  a  pump  for 
compressing  air  is  attached  to  it  by  a  rubber  tube.  Instead  of  this 
pump,  water  pressure  may  be  used  indirectly  by  attaching  a  strong 
bottle  to  the  water  supply  and  allowing  it  to  fill  slowly  with  water, 
and  at  the  same  time  to  force  out  the  air  through  a  tube  connected 
with  the  filtering  apparatus.  For  this  purpose  the  bottle,  having  a 
capacity  of  a  quart  or  more,  should  be  provided  with  a  rubber  stop- 
per through  which  two  short  tubes  are  passed.  One  of  these  is  con- 
nected with  the  water  supply  and  the  other  with  the  filter.  Of 
course  this  is  only  practicable  when  a  water  supply  with  sufficient 
pressure  is  available. 


FIG.  32. 


As  a  rule,  filtration  cannot  be  substituted  with  advantage  for  ster- 
ilization by  heat  in  the  preparation  of  culture  media.  Albuminous 
liquids  pass  through  the  filter  with  difficulty,  and  the  process  of 
sterilization  by  discontinued  heating  will  usually  prove  more  satis- 
factory than  filtration,  which  requires  extreme  precautions  to  pre- 
vent accidental  contamination  of  the  filtered  liquid.  Moreover,  the 
filter  may  change  the  composition  of  the  medium  passed  through  it 
by  preventing  the  passage  of  colloid  and  albuminous  material  in  so- 
lution. Thus,  in  an  attempt  to  separate  blood  corpuscles  from  the 
serum  by  filtration  through  a  Chamberland  filter,  the  writer  obtained 
a  transparent  liquid  which  did  not  coagulate  by  heat — i.  e. ,  the  albu- 
minous constituents  of  the  serum  did  not  pass  through  the  filter. 


VII. 
CULTURES   IN   LIQUID   MEDIA. 

PRIOR  to  the  introduction  of  gelatinous  media  by  Koch  in  1881, 
cultures  were  made  in  various  organic  liquids,  and  these  are  still 
largely  used,  being  for  certain  purposes  preferable  to  solid  media. 
The  method  of  preparing  and  sterilizing  the  flesh  infusions  and 
other  organic  liquids  commonly  used  has  already  been  given.  We 
are  here  concerned  with  the  various  modes  of  using  these  nutritive 
liquids  in  cultivating  bacteria. 

Flasks  and  tubes  of  various  forms  have  been  employed  by  differ- 
ent investigators,  but  the  most  useful  receptacle  for  liquid  as  well  as 
for  solid  culture  media  is  the  ordinary  test  tube.  These  are  care- 
fully cleaned,  plugged  with  a  cotton  air  filter,  sterilized  in  the  hot-air 
oven  at  150°  C.,  and  are  then  ready  to  receive  the  filtered  liquid. 
Usually  the  tube  should  not  be  filled  to  more  than  one-third  to  one- 
half  of  its  capacity.  Sterilization  of  the  culture  liquid  is  then  effected 
by  placing  the  tubes  in  the  steam  sterilizer  for  half  an  hour  on  three 
successive  days.  Before  using,  the  tubes  should  be  placed  for  a  few 
days  in  an  incubating  oven  at  30°  to  35°  C.  to  test  the  sterilization. 
This  is  especially  important  with  liquid  media,  for  if  a  single  living 
spore  is  present  it  may  give  rise  to  an  abundant  progeny,  which  will 
be  distributed  through  the  liquid  in  association  with  the  species 
which  has  been  planted.  In  solid  cultures,  on  the  contrary,  such  a 
spore  would  give  rise  to  a  colony,  which  by  its  locality  and  characters 
of  growth  would  probably  be  recognized  as  different  from  the  species 
planted,  and  consequently  accidental.  This  is  the  great  danger  in 
the  use  of  liquid  media  ;  imperfect  sterilization,  or  accidental  contami- 
nation by  atmospheric  germs,  may  lead  the  inexperienced  student 
into  serious  errors  resulting  from  the  assumption  that  the  micro- 
organisms present  in  his  cultures  are  all  derived  from  the  seed  he 
planted. 

On  the  other  hand,  liquid  media  are  more  convenient  than  solid 
when  it  is  the  intention  to  isolate  by  filtration  the  soluble  products  of 
bacterial  growth;  for  injection  into  animals  to  test  pathogenic  power; 
for  experiments  on  the  germicidal  or  antiseptic  power  of  chemical 
agents,  etc. 


CULTURES   IX   LIQUID    MEDIA.  63 

For  larger  quantities  of  liquid  than  can  be  held  in  an  ordinary 
test  tube  the  small  flasks  with  a  flat  bottom,  known  as  Erlenmeyer 
flasks,  are  very  convenient  (Fig.  33). 

In  his  earlier  researches  Pasteur  used  flasks  and  tubes  of  various 
forms,  which  served  a  useful  purpose,  but  have  been  displaced  in  his 
laboratory  by  the  simpler  form  of  apparatus  shown  in  Fig.  34. 
This  is  a  little  flask  having  a  cover  which  is  ground  to  fit  the  neck. 
This  cover  is  drawn  out  above  into  a  narrow  tube  which  admits 
oxygen  to  the  flask  through  a  cotton  air  filter.  To  obtain  access 
to  the  interior  of  the  flask  for  the  purpose  of  introducing  bacteria 
to  start  a  culture,  or  to  obtain  material  for  microscopical  examina- 
tion, the  cover  is  detached  at  the  ground  joint  by  a  gentle  twisting 
motion. 

There  is  much  less  danger  that  a  sterile  culture  liquid  will  become 


FIG.  33. 


FIG.  34. 


contaminated  during  the  momentary  removal  of  the  cover  from 
one  of  these  little  flasks,  or  of  the  cotton  plug  from  a  test  tube,  than 
is  usually  supposed.  Abundant  laboratory  experience  demonstrates 
that  such  contamination  by  bacteria  floating  in  the  atmosphere  rarely 
occurs.  The  spores  of  mould  fungi  are  commonly  more  abundant 
in  the  air,  but  even  these  do  not  very  frequently  fall  into  the  culture 
liquid  when  the  tube  is  opened  to  inoculate  it  with  the  bacteria  it  is 
proposed  to  cultivate.  This  inoculation  is  best  made  with  a  platinum 
wire,  bent  into  a  loop  at  the  free  extremity,  and  sealed  fast  into  the 
end  of  a  glass  rod  (Fig.  35).  This  is  sterilized  in  the  flame  of  a 
Bunsen  burner  or  alcohol  lamp  by  bringing  the  platinum  wire  to  a 
red  heat  and  passing  the  end  of  the  glass  rod  which  carries  it 
through  the  flame  several  times.  With  this  instrument  we  may 
transfer  a  little  drop  from  a  culture  to  the  sterile  fluid  in  another 


64 


CULTURES   IX   LIQUID   MEDIA. 


tube  for  the  purpose  of  starting  a  new  culture.  Or  we  may  start  a 
pure  culture  from  a  drop  of  blood  taken  from  the  veins  of  an  animal 
which  has  been  inoculated  with  anthrax,  or  any  similar  infectious 
disease  in  which  the  blood  is  invaded  by  a  bacterial  parasite. 

But  if  we  have  not  a  pure  culture  to  start  with  our  liquid  media 
do  not  afford  us  the  means  of  obtaining  one  ;  and  if  two  or  more 
bacteria  which  resemble  each  other  in  their  morphology  are  associated 
in  such  a  culture  we  cannot  differentiate  them,  and  are  likely  to  infer 
that  we  have  a  pure  culture  of  a  single  microorganism  when  this  is 
not  really  the  case. 

But  if  we  have  pure  stock  to  start  with  we  may  maintain  pure 
cultures  in  liquid  media  without  any  special  difficulty. 

Various  characters  of  groivth,  etc.,  are  to  be  observed  in  culti- 
vating different  microorganisms  in  liquid  media.  Thus  some  grow 
at  the  surface  in  the  form  of  a  thin  film  or  membranous  layer — "  my- 
coderma  "  —while  others  are  distributed  uniformly  through  the  liquid, 
rendering  it  opalescent  or  more  or  less  milky  and  opaque  ;  others, 
again,  form  little  flocculi  which  are  suspended  in  the  transparent 


FIG.  35. 


fluid.  Usually,  when  active  growth  has  ceased,  the  bacteria  fall 
the  bottom  of  the  tube  as  a  more  or  less  abundant,  white  or  colored, 
pulverulent  or  glutinous  deposit.  In  some  cases  the  liquid  is  colored 
with  a  soluble  pigment  formed  during  the  growth  of  the  bacteria, 
and  usually  this  is  formed  most  abundantly  at  the  surface,  where 
there  is  free  access  of  oxygen.  The  reaction  of  the  medium  is  often 
changed  as  a  result  of  the  growth  of  bacteria  in  it.  From  being  neu- 
tral it  may  become  decidedly  alkaline  or  acid  in  its  reaction.  These 
changes  may  be  observed  by  adding  a  litmus  solution  before  sterili- 
zation of  the  culture  medium,  and  observing  the  change  of  color 
when  an  acid-producing  bacterium  is  under  cultivation.  The  rt 
ducing  power  of  bacteria  upon  various  aniline  colors  may  also  be 
studied  ;  also  their  power  to  break  up  various  organic  substances,  as 
shown  by  the  evolution  of  gas  or  other  volatile  products  which 
may  be  collected,  or  by  substances  which  remain  in  solution  and 
can  be  studied  by  ordinary  chemical  methods. 

Drop  Cultures. — When  we  desire  to  study  the  life  history  of 
microorganism  and  to  witness  its  development  from  spores,  for  ex- 
ample, its  motions,  etc.,  the  method  of  cultivation  in  a  hanging  dro] 


CULTURES   IX    LIQUID    MEDIA.  65 

of  culture  fluid,  attached  to  a  thin  glass  cover  and  suspended  over  a 
circular  excavation  ground  out  of  a  glass  slide,  is  very  useful. 
Such  a  drop  culture  may  be  left  under  the  microscope  and  kept 
under  observation  for  hours  or  days. 

In  making  these  drop  cultures  it  is  necessary  to  sterilize  the  glass 
slides  and  thin  glass  covers  by  heat,  and  to  take  every  precaution  to 
prevent  the  inoculation  of  the  drop  of  culture  liquid  with  any  other 
bacteria  than  those  which  are  to  be  studied. 

The  simplest  form  of  moist  chamber  for  drop  cultures  consists  of 
an  ordinary  glass  slide  having  a  concave  depression,  about  fifteen 
millimetres  in  diameter,  ground  out  in  its  centre.  This  and  the  thin 
glass  cover,  having  been  sterilized  by  exposure  in  the  hot-air  oven  at 
150°  C.  for  an  hour  or  more,  or  by  passing  them  through  the  flame 
of  an  alcohol  lamp,  are  ready  for  use.  The  cover  glass  is  held  in 
sterile  forceps,  and  a  little  drop  of  the  culture  fluid  containing  the 
bacterium  to  be  studied  is  transferred  to  its  centre  by  means  of  the 
platinum  loop  heretofore  described.  It  is  best  to  spread  the  drop 
out  as  thin  as  possible,  and  it  may  be  inoculated,  from  a  pure  cul- 


FIG.   36. 


ture,  with  a  platinum  needle  (Fig.  36)  after  it  has  been  placed  upon 
the  cover.  This  is  then  inverted  over  the  hollow  place  in  the  glass 
slide,  and  it  is  customary  to  prevent  the  entrance  of  air  and  attach 
the  cover  by  spreading  a  little  vaseline  around  the  margin  of  the 
excavation. 

Another  form  of  moist  chamber  is  made  by  attaching  a  glass 
ring,  having  parallel,  ground  surfaces,  to  the  centre  of  a  glass  slide 
by  a  suitable  cement. 

In  Ranvier's  moist  chamber  there  is  a  central  eminence  sur- 
rounded by  a  groove  ground  into  the  glass  slide,  and  the  drop  of 
culture  fluid  is  in  contact  with  a  polished  glass  surface  below  as  well 
as  above.  This  affords  a  more  satisfactory  view  under  the  micro- 
scope. 

The  Author's  Culture  Method. — In  a  paper  read  at  the  meeting 
of  the  American  Association  for  the  Advancement  of  Science,  in 
August,  1881,  the  writer  described  a  method  of  conducting  culture 
experiments  which  he  has  since  used  extensively  and  with  very  satis- 
factory results.  The  liquid  culture  medium  is  preserved  in  little  flasks 
having  a  long  neck  which  is  hermetically  sealed.  The  principal  ad- 
vantages connected  with  the  use  of  these  little  flasks,  or  * '  Stern- 
5 


6G 


CULTURES   IN   LIQUID    MEDIA. 


berg's  bulbs,"  as  they  are  sometimes  called,  are  that  a  culture  me- 
dium may  be  preserved  in  them  indefinitely  and  that  they  are  easily 
transported  from  place  to  place;  whereas  test  tubes,  Pasteur's  flasks, 
and  similar  receptacles  must  be  kept  upright,  and  after  a  time  the 
culture  liquid  in  them  is  changed  in  its  composition  by  evaporation. 
They  are  also  liable  to  be  contaminated  by  the  entrance  of  mould 
fungi  when  kept  in  a  damp  place.  The  spores  of  these  fungi,  falling 
upon  the  surface  of  the  cotton  air  filter,  germinate,  and  the  myce- 
lium grows  down  through  the  cotton  into  the  interior  of  the  tube, 
where  a  new  crop  of  spores  is  quickly  formed.  It  is,  therefore,  a 
convenience  to  have  sterile  culture  liquids  always  ready  for  use  in 
a  receptacle  which  can  be  packed  in  a  box  and  transported  from 
place  to  place  ;  but  for  every-day  use  in  the  laboratory  the  ordinary 


FIG.  37, 

test  tube,  with  its  cotton  air  filter,  is  the  most  economical  and  conve- 
nient receptacle  for  culture  liquids  as  well  as  for  solid  media.  With 
reference  to  the  method  of  making  and  using  these  little  flasks,  I 
quote  from  a  paper  published  in  the  American  Journal  of  the 
Medical  Sciences  in  1883  :' 

The  culture  flasks  employed  contain  from  one  to  four  fluidrachms. 
They  are  made  from  glass  tubing-  of  three-  or  four  tenths  inch  diameter,  and 
those  which  the  writer  has  used  in  his  numerous  experiments  have  all  been 
"  home-made."  It  is  easier  to  make  new  flasks  than  to  clean  old  ones,  and 
they  are  thrown  away  after  being  once  used.  Bellows  operated  by  foot,  and 
a  flame  of  considerable  size — gas  is  preferable — will  be  required  by  one  who 
proposes  to  construct  these  little  flasks  for  himself.'2  After  a  little  practice 
they  are  made  rapidly;  but  as  a  large  number  are  required,  the  time  and 
labor  expended  in  their  preparation  are  no  slight  matter.  After  blowing  a 
bulb  at  the  extremity  of  a  long  glass  tube,  of  the  diameter  mentioned,  this 
is  provided  with  a  slender  neck,  drawn  out  in  the  flame,  and  the  end  of  this 

1  "  The  Germicide  Value  of  Certain  Therapeutic  Agents,"  op.  cit.,  vol.  clxx. 
8  A  glass-blower  ought  to  make  them  for  two  or  three  dollars  per  hundred. 


CULTURES   IN   LIQUID   MEDIA.  67 

is  hermetically  sealed.  Thus  one  little  flask  after  another  is  made  from  the 
same  piece  of  tubing  until  this  becomes  too  short  for  further  use.  To  intro- 
duce a  culture  liquid  into  one  of  these  little  flasks,  heat  the  bulb  slightly, 
break  off  the  sealed  extremity  of  the  tube  and  plunge  it  beneath  the  surface 
of  the  liquid  (Fig.  37).  The  quantity  which  enters  will  of  course  depend 
upon  the  heat  employed  and  the  consequent  rarefaction  of  the  enclosed  air. 
Ordinarily  the  bulb  is  filled  to  about  one-third  of  its  capacity  with  the  cul 
ture  liquid,  leaving  it  two  thirds  full  of  air  for  the  use  of  the  microscopic 
plants  which  are  to  be  cultivated  in  it.  ...  Sterilization  is  effected  by  heat 
after  the  liquid  has  been  introduced  and  the  neck  of  the  flask  hermetically 
sealed  in  the  flame  of  an  alcohol  lamp. 

Sterilization  may  be  effected  by  boiling  for  an  hour  in  a  bath  of  paraffin 
or  of  concentrated  salt  solution,  by  which  a  temperature  considerably  above 
that  of  boiling  water  is  secured.  The  writer  is  in  the  habit  of  preparing  a 
considerable  number  of  these  flasks  at  one  time,  and  leaving  them,  in  a  suit- 
able vessel  filled  with  water,  for  twenty  four  hours  or  longer  011  the  kitchen 
stove.1 

To  inoculate  the  liquid  contained  in  one  of  these  little  flasks  with  mi- 
croorganisms from  any  source,  the  end  of  the  tube  is  first  heated  to  destroy 
germs  attached  to  the  exterior;  the  extremity  is  then  broken  off  with  steril- 
ized (by  heat)  forceps;  the  bulb  is  very  gently  heated,  so  as  to  force  out  a 
little  air,  and  the  open  end  is  plunged  into  the  liquid  containing  the  organ- 
ism to  be  cultivated  (or  into  a  vein,  or  one  of  the  solid  viscera  of  an  animal 
dead  from  an  infectious  germ  disease,  such  as  anthrax). 

Inoculation  from  one  tube  to  another  may  also  be  effected  by  means  of 
the  ordinary  platinum  wire  needle. 

Before  the  introduction  of  Koch's  plate  method  for  isolating  bac- 
teria in  pure  cultures,  certain  methods  had  been  proposed,  and  em- 
ployed to  some  extent,  which  at  present  have  a  historical  value  only. 

Thus  Klebs  (1873)  proposed  to  take  from  a  first  culture  in  which 
two  or  more  species  were  associated  a  minute  quantity,  by  means  of  a 
capillary  tube,  and  with  this  to  inoculate  a  second  culture.  By  re- 
peating this  procedure  several  times  he  expected  to  exclude  all  except 
the  species  which  was  present  in  the  greatest  abundance  and  which 
multiplied  most  rapidly  in  the  medium  employed. 

The  method  by  dilution,  first  employed  with  precision  by  Brefeld 
(1872)  in  obtaining  pure  cultures  of  mould  fungi,  and  subsequently 
by  Lister  for  the  isolation  of  bacteria,  consists  in  so  diluting  a  minute 
quantity  of  the  mixed  culture  that  the  number  of  bacteria  in  the  dilu- 
tion may  be  less  than  one  for  each  drop  of  the  liquid.  If  now  a 
single  drop  be  added  to  each  of  a  series  of  tubes  containing  a  small 
quantity  of  sterile  bouillon,  some  of  the  inoculations  made  may  give 
a  pure  culture,  as  the  drop  may  have  contained  but  a  single  vege- 
tative cell. 

Another  method  of  obtaining  a  pure  culture  in  liquid  media,  when 
several  microorganisms  are  associated  which  have  a  different  ther- 

1  Where  a  steam  sterilizer  is  at  hand  they  will  be  most  conveniently  sterilized  in 
the  usual  way,  by  subjecting  them  to  the  boiling  temperature  for  an  hour  at  a  time 
on  three  successive  days. 


68 


CULTURES    IX    LIQUID    MEDIA. 


mal  death-point,  consists  in  the  application  of  heat  and  thus  destroy- 
ing all  except  the  most  resistant  species.  This  method  is  especially 
applicable  when  one  of  the  species,  only,  forms  spores.  By  subject- 
ing the  mixed  culture  to  a  temperature  which  is  sufficient  to  destroy 
all  the  vegetative  cells  in  it,  the  more  resistant  spores  are  left  and, 

under  favorable  conditions,  may  subsequently 
vegetate  and  give  us  a  pure  culture  of  the 
species  to  which  they  belong. 

Fermentation. — The  development  of  certain 
bacteria  is  attended  with  an  evolution  of  gas, 
especially  in  media  containing  grape  sugar  or 
glycerin.  For  the  determination  of  the  quantity 
and  kind  of  gas  produced  by  a  given  micro- 
organism the  fermentation  tube  recommended 
by  Theobald  Smith  has  special  advantages. 
This  is  a  bent  tube  (Eihorn's)  supported  upon 
a  glass  base  as  shown  in  the  accompanying 
figure  taken  from  the  catalogue  of  Eimer  & 
Amend.  The  graduation  shown  upon  the  up- 
right arm  is  not  essential  for  ordinary  labora- 
tory work.  A  liquid  culture  medium  containing 
one  to  two  per  cent  of  grape  sugar  is  usually 
used.  This  is  introduced  into  the  upright  arm 
of  the  fermentation  tube,  where  it  is  held  by  atmospheric  pressure. 
A  cotton  plug  is  placed  in  the  opening  of  the  short  and  bulbous  arm 
of  the  tube,  which  is  intended  as  a  receptacle  for  the  culture  liquid 
when  it  is  forced  out  of  the  closed  arm  by  the  accumulation  of  gas  at 
its  upper  extremity. 


FIG.  38. 


VIII. 

CULTURES  IX  SOLID  MEDIA. 

THE  introduction  of  solid  culture  media  in  1881  by  the  famous 
German  bacteriologist,  Robert  Koch,  inaugurated  a  new  era  in  the 
progress  of  our  knowledge  relating  to  the  bacteria.  His  methods 
enable  us  to  obtain  pure  cultures  with  ease  and  certainty,  and  to 
study  the  morphological  and  biological  characters  of  each  species 
free  from  the  complications  which  led  to  so  much  error  and  confusion 
before  these  methods  were  introduced.  We  have  already  given  an 
account  of  the  method  of  preparing  and  sterilizing  the  various  solid 
culture  media,  and  are  here  concerned  with  the  manner 
in  which  they  are  used  and  the  special  advantages  which 
they  afford. 

Koch's  flesh-peptone-gelatiii,  which  contains  ten  per 
cent  of  gelatin,  is  a  transparent  jelly  which  liquefies  at 
from  22°  to  24°  C.     It  is  a  favorable  culture  medium  for 
a  great  number  of  bacteria,  and  many  species  show  de- 
finite characters  of  growth  in  this  medium  which  serve  to 
differentiate  them.     One  of  the  most  prominent  of  these 
characters  depends  upon  the  fact  that  some  bacteria  liquefy 
gelatin  and  others  do  not.     This  is  made  apparent  when 
we  make  "stab  cultures."     This  is  the  usual  manner  of 
inoculating  a  solid  culture  medium,  and  is  illustrated  in 
Fig.   39.     A  platinum  needle,   consisting  of  a   piece  of 
platinum  wire  inserted  into  a  glass  rod  which  serves  as  a 
handle,  is  passed  through  the  flame  of  an  alcohol  lamp  to 
sterilize  it.      When  cooled,  which  occurs  very  quickly,  the 
point  is  introduced  into  the  material  containing  the  bac- 
teria to  be  planted  in  the  gelatin  medium.     We  may  ob- 
tain our  seed  for  a  pure  culture  from  a  single  colony,  from         FIG.  39. 
another  stab  culture,  from  the  blood  of  an  infected  animal, 
etc.     The  point  of  the  needle  is  then  carried  into  the  sterilized  jelly, 
as  shown  in  the  figure,  care  being  taken  to  introduce  it  in  the  central 
line  and  in  a  direction  parallel  with  the  sides  of  the  tube.     It  is  best 


70 


CULTURES   IN   SOLID   MEDIA. 


always  to  hold  the  tube  inverted  during  the  inoculation,  and  not 
remove  the  cotton  air  filter  until  we  are  ready  to  make  it.  The 
cotton  plug  is  then  returned  to  its  place  and  the  platinum  needle 
again  brought  to  a  red  heat  to  destroy  any  bacteria  which  remain 
attached  to  it. 

Sometimes  it  is  an  advantage  to  have  the  culture  medium  with  a 


FIG.  40. 


sloping  surface,  as  shown  in  Fig.  40.     We  may  then  draw  the 

die  over  the  surface  in  a  longitudinal  direction,  and  by  this  means 

distribute  the  seed  in  a  line  along  which  development  will  take  place. 

The  characters  of  growth  in  these  stab  cultures  in  gelatin  are 


very  various.  Non-liquefying  bacteria  may  grow  only  on  the  sui 
face,  as  at  a,  Fig.  40A ;  or  both  on  the  surface  and  along  the  line 
of  puncture,  as  at  b;  or  only  at  the  bottom,  as  at  c.  In  the  first 
case  the  microorganism  is  aerobic — that  is,  it  requires  oxygen,  and 
grows  only  in  the  presence  of  this  gas.  In  the  second  case  it  is 
not  strictly  aerobic,  but  may  grow  either  in  the  presence  of  oxygen 


CULTURES   IN   SOLID   MEDIA. 


71 


or  in  its  absence — a  facultative  anaerobic.  In  the  third  case  the 
microorganism  is  an  anaerobic,  which  cannot  grow  in  the  presence 
of  oxygen,  and  consequently  does  not  grow  upon  the  surface  of  the 
culture  medium  or  along  the  upper  portion  of  the  line  of  puncture. 

Again,  we  have  differences  as  to  the  character  of  growth  upon  the 
surface  or  along  the  line  of  puncture.  The  surface  growth  may  be 
a  little  mass  piled  up  at  the  point  where  the  needle  entered  the  gela- 
tin ;  or  it  may  form  a  layer  over  the  entire  surface,  and  this  may 
be  thin  or  thick,  dry  or  moist,  viscid  or  cream-like,  and  of  various 
colors — green,  blue,  red,  or  yellow,  of  different  shades — or  more  fre- 
quently of  a  milk-white  color. 


a 


The  growth  along  the  line  of  puncture  also  differs  greatly  with 
different  species.  We  may  have  a  number  of  scattered  spherical 
colonies  (a,  Fig.  41),  and  these  may  be  translucent  or  opaque  ;  or  we 
may  have  little  tufts,  like  moss,  projecting  from  the  line  of  puncture 
(&,  Fig.  41)  ;  or  slender,  filamentous  branches  may  grow  out  into  the 
gelatin  (c,  Fig.  41). 

The  liquefying  bacilli  also  present  different  characters  of  growth. 
Thus  liquefaction  may  take  place  all  along  the  line  of  puncture, 
forming  a  long  and  narrow  funnel  of  liquefied  gelatin  (a,  Fig.  42)  ; 
or  we  may  have  a  broad  funnel,  as  at  b  ;  or  a  cup-shaped  cavity,  as 
at  c;  or  the  upper  liquefied  portion  may  be  separated  from  that 
which  is  not  liquefied  by  a  horizontal  plane  surface,  as  at  d. 


CULTURES   IN    SOLID    MEDIA. 


The  characters  of  growth  in  agar-agar  jelly  are  not  so  varied, 
but  this  medium  possesses  the  advantage  of  not  liquefying  at  a  tem- 
perature of  35°  to  38°  C.,  which  is  required  for  the  development  of 
certain  pathogenic  bacteria.  Variations  in  mode  of  growth  are 
also  manifested  in  nutrient  agar  similar  to  those  referred  to  as  pro- 
duced by  non-liquefying  bacteria  in  flesh-peptone-gelatin.  These 
relate  to  the  surface  growth  and  to  growth  along  the  line  of  punc- 
ture. One  character  not  heretofore  mentioned  consists  in  the  for- 
mation of  gas  bubbles  in  stab  cultures  either  in  gelatin  or  agar. 

Colonies. — If  we  melt  the  gelatin  or  agar  in  a  test  tube,  pour 
the  liquid  medium  into  a  shallow  glass  dish  previously  sterilized, 


FIG.  42. 

and  allow  it  to  cool  while  properly  protected  by  a  glass  cover,  we 
will  have  a  broad  surface  of  sterile  nutrient  material.  If  now  we  ex- 
pose it  to  the  air  for  ten  or  fifteen  minutes,  and  again  cover  it  and 
put  it  aside  for  two  or  three  days  at  a  favorable  temperature,  we  can 
scarcely  fail  to  have  a  number  of  colonies  upon  the  surface  of  the 
culture  medium,  which  have  been  developed  from  atmospheric  germs 
which  were  deposited  upon  it  during  the  exposure.  Each  of  these 
colonies,  as  a  rule,  is  developed  from  a  single  bacterium  or  spore, 
and  consequently  the  little  mass,  visible  to  the  naked  eye,  which  we 
call  a  colony,  is  a  pure  culture  of  a  particular  species.  In  this  ex- 
periment we  are  more  apt  to  have  colonies  of  mould  fungi  than  of 
bacteria,  but  the  principle  is  the  same,  viz. ,  that  a  colony  developed 
from  a  single  germ  is  a  pure  culture.  By  touching  our  platinum 


CULTURES   IX   SOLID   MEDIA.  73 

needle,  then,  to  such  a  colony,  which  is  quite  independent  of,  and 
well  separated  from,  all  others,  we  may  make  a  stab  culture  in  gela- 
tin or  agar,  and  preserve  the  pure  culture  for  further  study.  This 
is  a  most  important  advantage  which  pertains  to  the  use  of  solid 
culture  media.  It  is  a  singular  fact  that,  as  a  rule,  colonies  of  bac- 
teria which  lie  near  each  other  do  not  grow  together,  but  each  re- 
mains distinct.  If  there  are  but  few  colonies,  each  one,  having1 

7  "  O 

plenty  of  room,  may  grow  to  considerable  size  ;  if  there  are  many 
and  they  are  crowded,  they  remain  small,  but  are  still  independent 
colonies. 

Now,  these  colonies  differ  greatly  in  their  appearance  and  char- 
acters of  growth,  according  to  the  species  (Fig.  43).  Some  are 
spherical,  and  these  may  be  translucent  or  opaque,  or  they  may  have 
an  opaque  nucleus  surrounded  by  a  transparent  zone.  Again,  the 


FIG.  43.— Colonies  of  Bacteria. 

outlines  may  be  irregular,  giving  rise  to  amoeba-like  forms,  or  to  a 
fringed  or  plaited  margin,  or  the  form  may  be  that  of  a  rosette,  etc.  ; 
or  the  colony  may  appear  to  be  made  up  of  overlapping  scales  or 
masses,  or  of  tangled  filaments ;  or  it  may  present  a  branching 
growth.  In  the  case  of  liquefying  bacteria,  when  the  colonies  have 
developed  in  a  gelatin  medium  they  commonly  do  not  at  once  cause 
liquefaction  of  the  gelatin,  but  at  the  end  of  twenty-four  hours  or 
more  the  gelatin  about  them  commences  to  liquefy  and  they  are 
seen  in  a  little  funnel  of  transparent  liquefied  gelatin ;  or  in  other 
cases  little  opaque  drops  of  liquefied  gelatin  are  seen,  which,  as  the 
liquefaction  extends,  run  together.  All  of  these  characters  are  best 
studied  under  a  low-power  lens,  with  an  amplification  of  five  to 
twenty  diameters  ;  an:!  by  a  careful  observation  of  the  differences  in 
the  form  and  development  of  colonies  we  are  greatly  assisted  in  the 
differentiation  of  species. 

Single,  isolated  colonies  do  not  always  contain  a  single  species, 
for  they  are  not  always  developed  from  a  single  cell.     We  may  have 


74  CULTURES   IN   SOLID   MEDIA. 

deposited  upon  our  plate,  exposed  as  above  described,  a  little  mass 
of  organic  material  containing  two  or  more  different  bacteria,  and 
this  would  serve  as  the  nucleus  of  a  colony  from  which  we  could  not 
obtain  a  pure  culture. 

Koch's  Plate  Method. — In  the  experiment  above  described, 
colonies  were  obtained  from  air-borne  germs  which  were  deposited 
upon  the  surface  of  our  gelatin  medium.  By  Koch's  famous  "  plate 
method  "  we  obtain  colonies  of  any  particular  microorganism  which 
we  desire  to  study,  or  of  two  or  more  associated  bacteria  which  we 
desire  to  study  separately  in  pure  cultures.  Evidently,  when  we 
have  obtained  separate  colonies  of  different  bacteria  upon  the  sur- 
face of  a  solid  culture  medium,  we  can  easily  obtain  a  pure  culture 
of  each  by  inoculating  stab  cultures  from  single  colonies. 

To  obtain  separate  colonies  we  resort  to  the  ingenious  method  of 
Koch.  Three  test  tubes  containing  a  small  quantity  of  nutrient 
gelatin  (or  of  agar)  are  commonly  employed.  The  tubes  are  num- 
b3red  1,  2,  and  3.  The  first  step  consists  in  liquefying  the  nutrient 
jelly  by  heat,  and  it  will  be  well  for  beginners  to  place  the  tubes  in 
a  water  bath  having  a  temperature  of  about  40°  C.  (104°  F.)  for  the 
purpose  of  keeping  the  culture  material  liquid,  and  at  the  same  time 
at  a  temperature  which  is  not  high  enough  to  destroy  the  vitality  of 
the  bacteria  which  are  to  be  planted.  We  next,  by  means  of  a 
platinum- wire  loop  or  the  platinum  needle  used  for  stab  cultures, 
introduce  into  tube  No.  1  a  small  amount  of  the  culture,  or  material 
from  any  source,  containing  the  bacteria  under  investigation.  Care 
must  be  taken  not  to  introduce  too  much  of  this  material,  and  it 
must  be  remembered  that  the  smallest  visible  amount  may  contain 
many  millions  of  bacteria.  The  reason  for  using  three  tubes  will 
now  be  apparent.  It  is  usually  impossible  to  introduce  a  few  bac- 
teria into  tube  No.  1,  but  we  effect  our  object  by  dilution,  as  follows  : 
With  the  platinum- wire  loop  we  take  up  a  minute  drop  of  the  fluid  in 
tube  No.  1,  through  which  the  bacteria  have  been  distributed  by 
stirring,  and  carry  it  over  to  tube  No.  2.  Washing  off  the  drop  by 
stirring,  we  may  repeat  this  a  second  or  third  time — this  is  a  matter 
of  judgment  and  experience ;  often  it  will  suffice  to  carry  over  a 
single  ose  (the  German  name  for  the  platinum- wire  loop).  Next 
we  carry  over  one,  or  two,  or  three  ose  from  tube  No.  2  to  tube  No. 
3.  By  this  procedure  we  commonly  succeed  in  so  reducing  the  num- 
ber of  bacteria  in  tube  No.  3  that  only  a  few  colonies  will  develop 
upon  the  plate  which  we  subsequently  make  from  it;  or  it  may  happen 
that  the  dilution  has  been  carried  too  far  and  that  no  colonies  de- 
velop upon  the  plate  made  from  this  tube,  in  which  case  we  are 
likely  to  get  what  we  want  from  tube  No.  2.  The  next  step  is  to 
pour  the  liquid  gelatin  upon  sterilized  glass  plates,  which  are  num- 


CULTURES   IN   SOLID    MEDIA.  75 

bered  to  correspond  with  the  tubes.  The  plates  used  by  Koch  are 
from  eight  to  ten  centimetres  wide  and  ten  to  twelve  centimetres 
long.  They  must  be  carefully  cleaned  and  sterilized  in  the  hot-air 
oven,  at  150°  C.,  for  two  hours.  They  may  be  wrapped  in  paper  be- 
fore sterilization,  or  placed  in  a  metal  box  especially  made  for  the 
purpose.  In  order  that  the  liquid  gelatin  may  be  evenly  distributed 
upon  the  plate  the  apparatus  shown  in  Fig.  44:  is  used.  This  con- 
sists of  a  glass  plate,  g,  supported  by  a  tripod  having  adjustable  feet. 
By  means  of  the  spirit  level  /  the  glass  plate  is  adjusted  to  a  hori- 
zontal position.  A  sterilized  glass  plate  is  placed  in  the  glass  tray, 
shown  in  the  figure,  and  the  gelatin  from  one  of  the  tubes  is  care- 
fully poured  upon  it  and  distributed  upon  its  surface  with  a  steril- 
ized glass  rod,  care  being  taken  not  to  bring  it  too  near  the  edge  of 
the  plate.  The  glass  tray  in  then  covered  until  the  gelatin  has 
cooled  sufficiently  to  become  solid,  after  which  plate  No.  1  is  re- 
moved and  plates  Nos.  2  and  3  are  made  in  the  same  way.  In 


FIG.  44. 

order  to  save  time  it  is  customary  to  fill  the  glass  tray  shown  in  the 
figure  with  ice  water,  to  place  a  second  glass  support  upon  it,  and 
upon  this  the  sterilized  glass  plate  upon  which  the  liquid  gelatin  is 
poured.  This  is  protected  by  a  glass  cover,  as  before,  until  the  gela- 
tin becomes  solid. 

The  three  plates,  prepared  as  directed,  are  put  aside  in  a  glass 
jar  of  the  form  shown  in  Fig.  44,  one  being  supported  above  the 
other  by  a  bench  of  sheet  zinc  or  glass. 

Petri's  Dishes. — A  modification  of  the  plate  method  of  Koch, 
which  has  some  advantages,  consists  in  the  use  of  three  small  glass 
dishes  of  the  same  form  as  the  larger  one  used  by  Koch  to  contain 
the  plates.  These  dishes  of  Petri  are  about  ten  to  twelve  centime- 
tres in  diameter  and  one  to  1.5  centimetres  high,  the  cover  being  of 
the  same  form  as  the  dish  into  which  the  gelatin  is  poured.  These 
dishes  take  less  room  in  the  incubating  oven  than  the  larger  glass 
jar  used  in  the  plate  method,  and  they  do  not  require  the  use  of  a 
levelling  apparatus.  The  colonies  also  may  be  examined  and 
counted,  if  desired,  without  removing  the  cover,  and  consequently 


76 


CULTURES   IX   SOLID    MEDIA. 


without  the  exposure  which  occurs  when  a  plate  prepared  by  Koch's 
method  is  under  examination. 

In  agar-agar  cultures  or  in  gelatin  cultures  of  non-liquefying 
bacteria  made  in  Petri's  dishes,  we  may  examine  and  count  colonies, 
without  removing  the  cover,  by  inverting  the  dish. 

In  pouring  the  liquefied  gelatin  from  the  test  tubes  in  which  the 
dilution  has  been  made  into  sterilized  Petri's  dishes,  care  must  be 
taken  to  first  sterilize  the  lip  of  the  test  tube  by  passing  it  through 
the  flame  of  a  lamp.  We  may  at  the  same  time  burn  off  the  top  of 
the  cotton  plug,  then  remove  the  remaining  portion  with  forceps, 
when  the  lip  has  cooled,  for  the  purpose  of  pouring  the  liquid  into  the 
shallow  dish. 

Von  Esmarch' s  Roll  Tubes. — Another  very  useful  modification 
of  Koch's  plate  method  is  that  of  von  Esmarch.  Instead  of  pouring 
the  liquefied  gelatin  or  agar  medium  upon  plates  or  in  shallow 


FIG.  45. 

dishes,  it  is  distributed  in  a  thin  layer  upon  the  walls  of  the  test  tube 
containing  it.  This  is  done  by  rotating  the  tube  upon  a  block  of  ice 
or  in  iced  water.  Esmarch  first  used  a  tray  containing  iced  water, 
and  to  prevent  the  wetting  of  the  cotton  filter  a  cap  of  thin  rubber 
was  placed  over  the  end  of  the  tube.  It  is  more  convenient  to  turn 
the  tubes  upon  a  block  of  ice  having  a  horizontal  flat  surface,  in 
which  a  shallow  groove  is  first  made  by  means  of  a  test  tube  con- 
taining hot  water  (Fig.  45).  Or,  in  the  winter,  we  may  turn  the 
tube  under  a  stream  of  cold  water  from  the  city  supply — i.  e. ,  from  a 
faucet  in  the  laboratory.  A  little  practice  will  enable  the  student  to 
distribute  the  culture  medium  in  a  uniform  layer  on  the  walls  of  the 
test  tube,  and  as  soon  as  it  is  quite  solidified  these  may  be  placed 
aside  for  the  development  of  colonies  from  the  bacteria  which  had 
been  introduced.  When  roll  tubes  are  made  from  the  agar  jelly  it  is 
best  to  place  the  tubes  in  a  nearly  horizontal  position,  for  if  placed 
upright  at  once  the  film  of  jelly  is  likely  to  slip  from  the  walls  of  the 


CULTURES   IX   SOLID    MEDIA.  «  « 

tube.  This  is  due  to  the  fact  that  a  little  fluid  is  pressed  out  of  the 
jelly,  probably  by  a  slight  contraction  while  cooling.  If  the  tubes 
are  slightly  inclined  from  the  horizontal  the  film  does  not  slip  and 
the  fluid  accumulates  at  the  bottom.  After  a  day  or  two  they  may 
be  placed  in  an  upright  position. 

These  roll  tubes  possess  several  advantages.  They  are  quickly 
made  and  take  but  little  space  in  the  incubating  oven,  and  the  film 
of  jelly  is  protected  from  contamination  by  atmospheric  germs. 
When  colonies  have  formed  we  may  examine  them  through  the  thin 
\valls  of  the  tube,  either  with  a  pocket  lens  or  a  low-power  objective. 
In  making  a  stab  culture  from  a  single  colony  in  one  of  these  roll 
tubes,  we  invert  the  tube,  remove  the  cotton  air  filter,  and  pass  the 
point  of  a  sterilized  platinum  needle  up  to  the  selected  colony.  In 
the  same  way  we  obtain  material  for  microscopical  examination. 

Streak  Cultures. — In  his  earlier  experiments  with  solid  culture 
media  Koch  made  "  streak  cultures  "  by  drawing  the  point  of  a  plati- 
num needle,  charged  with  bacteria,  over  the  surface  of  a  gelatin  or 
agar  plate  ;  and  this  method  is  still  useful  in  certain  cases.  If  we 
draw  the  needle  over  the  moist  surface  several  times  in  succession 
the  greater  number  of  bacteria  will  be  deposited  in  the  first  streak, 
and  in  the  second  or  third  single  cells  are  likely  to  be  left  at  such 
intervals  from  each  other  that  each  will  develop  an  independent 
colony.  If  the  streaks  were  made  with  impure  stock  we  may  thus 
succeed  in  getting  separate  colonies  of  the  several  bacteria  contained 
in  it,  so  that  this  method  may  be  employed  for  obtaining  pure  cul- 
tures. But  for  this  purpose  it  is  much  inferior  to  the  plate  method, 
and  it  is  chiefly  used  for  observing  the  growth  of  bacteria  on  the  sur- 
face of  solid  culture  media.  Thus  we  commonly  make  a  streak  upon 
the  surface  of  cooked  potato  or  solidified  blood  serum  in  studying  the 
development  of  various  bacteria  on  these  culture  media. 

Cultures  upon  Blood  Serum. — The  use  of  blood  serum  as  a 
solid  medium  is  practically  restricted  to  stab  cultures  and  streak 
cultures,  for  we  cannot  substitute  it  for  the  gelatin  and  agar  media 
in  making  plates  and  roll  tubes.  This  is  because  it  only  becomes  solid 
at  a  temperature  which  would  be  fatal  to  most  bacteria  (70°  C.),  and 
when  once  made  solid  by  heat  cannot  again  be  liquefied.  Its  use  is, 
therefore,  restricted  mainly  to  the  cultivation  of  bacteria  for  which 
it  is  an  especially  favorable  medium.  It  may  be  used,  however,  in 
combination  with  a  gelatin  or  agar  medium.  For  this  purpose  it  is 
most  conveniently  kept  in  a  fluid  condition  in  the  little  flasks  hereto- 
fore described  ("  Sternberg's  bulbs  "). 

The  gelatin  or  agar  jelly  in  test  tubes  is  liquefied  by  heat  and 
cooled  in  a  water  bath  to  about  40°  C.  The  desired  amount  of  ste- 
rile blood  serum  is  then  forced  into  each  tube  by  passing  the  slender 


rvo 
<  o 


CULTURES   IN   SOLID    MEDIA. 


neck  of  the  little  flask  along  the  side  of  the  cotton  filter  (see  Fig.  46) 
and  applying  gentle  heat  to  the  bulb.  The  slender  neck  is  first  ste- 
rilized by  passing  it  through  a  flame,  and  the  point  is  broken  off 
with  sterile  forceps.  After  inoculating  the  liquefied  medium  in  the 
test  tubes  in  the  usual  manner  we  may  make  plates  or  roll  tubes. 

Cultures  on  Cooked  Potato. — The  method  of  preparing  pota- 
toes for  surface  cultures  has  already  been  given  (page  48).  It  was 
in  using  them  that  Koch  first  got  his  idea  of  the  importance  of  solid 
media,  which  led  to  his  introduction  of  the  use  of  gelatin  and  agar- 
agar  and  the  invention  of  the  plate  method.  By  means  of  streak 


Fin.  46. 

cultures  upon  potato  he  had  succeeded  in  obtaining  isolated  colonies 
and  pure  cultures.  We  now  use  the  potato  chiefly  for  the  purpose 
of  differentiating  species.  Some  bacteria  grow  on  the  surface  of 
cooked  potato  and  some  do  not.  Those  which  do  present  various 
characters  of  growth.  Thus  we  have  differences  as  to  color,  as  to 
rapidity  of  growth,  as  to  the  character  of  the  mass  formed — thick 
or  thin,  viscid,  moist  or  dry,  restricted  to  line  of  inoculation  or  ex- 
tending over  the  entire  surface,  etc. 

Instead  of  using  a  cut  section  of  the  potato  in  the  manner  here- 
tofore described,  we  may  make  a  puree  by  mashing  the  peeled  and 
cooked  tubers  and  distributing  the  mass  in  Erlenmeyer  flasks.  After 


CULTURES   IN   SOLID    MEDIA.  79 

thorough  sterilization  by  steam  the  culture  medium  is  ready  for  use. 
In  the  same  way  other  vegetables,  or  bread,  etc. ,  may  be  used  for 
special  purposes,  and  especially  for  cultures  of  the  mould  fungi. 

Potatoes  usually  have  a  slightly  acid  reaction,  and  on  this  ac- 
count certain  bacteria  will  not  grow  upon  them.  This  acid  reaction 
is  not  constant  and  differs  in  degree,  and  as  a  result  we  may  have 
decided  differences  in  the  growth  of  the  same  species  upon  different 
potatoes.  To  overcome  this  objection  the  writer  has  sometimes  neu- 
tralized the  cones  of  potato  in  test  tubes  (see  Fig.  21,  page  49)  by 
first  boiling  them  in  water  containing  a  little  carbonate  of  soda. 
The  liquid  is  poured  off  after  they  have  been  in  the  steam  sterilizer 
for  half  an  hour,  and  they  are  returned  for  sterilization. 

Salomonson's  Method  of  cultivation  in  capillary  tubes  has  a  his- 
torical value  only  since  the  introduction  of  Koch's  plate  method. 

The  following  modifications  of  Koch's  plate  cultures  have  recently 
been  introduced: 

Kruse  (1894)  pours  the  liquefied  gelatin  or  agar  into  Petri  dishes, 
and  after  it  is  solidified  brushes  the  surface  with  a  sterilized  camel's- 
hair  brush  which  has  been  dipped  into  water  containing  in  suspen- 
sion— properly  diluted — the  bacteria  to  be  studied.  By  this  procedure 
surface  colonies  only  are  obtained.  Von  Freudenreich  (1894)  prefers 
to  pour  the  contents  of  the  test  tube  upon  the  surface  of  the  sterile 
medium,  in  Petri  dishes.  The  fluid  is  allowed  to  run  off  by  placing 
the  Petri  dish  in  a  vertical  position,  and  this  is  subsequently  placed  in 
the  incubating  oven  in  an  inverted  position — i.e.,  with  cover  below. 
To  obtain  satisfactory  plates  with  well-separated,  superficial  colonies 
it  may  be  necessary  to  use  two  or  three  dilutions,  made  in  sterilized 
water  in  the  usual  way — i.e.,  from  one  tube  to  another,  by  means  of 
the  platinum  wire  having  a  loop  at  its  extremity. 


IX. 


CULTIVATION"  OF  ANAEROBIC  BACTERIA. 

PASTEUR  (1861)  .first  pointed  out  the  fact  that  certain  species  of 
bacteria  not  only  grow  in  the  entire  absence  of  oxygen,  but  that  for 
some  no  growth  can  occur  in  the  presence  of  this  gas.  Such  bacteria 
are  found  in  the  soil,  and  in  the  intestines  of  man  and  the  lower  ani- 
mals. The  cultivation  of  ''strict  anae'robics "  calls  for  methods  by 
which  oxygen  is  excluded.  The  "facultative  anae'robics''  grow 


FIG.  47 


FIG.  48. 


either  in  the  presence  or  absence  of  oxygen.  There  are  various  gra- 
dations in  this  regard,  from  the  strictly  aerobic  species  which  re- 
quire an  abundance  of  oxygen  and  will  not  grow  in  its  absence,  to 
the  strictly  anaerobic  species  which  will  not  grow  if  there  is  a  trace 
of  oxygen  in  the  medium  in  which  we  propose  to  cultivate  them. 
Among  the  most  interesting  pathogenic  bacteria  which  are  strictly 
anaerobic  are  the  bacillus  of  tetanus,  the  bacillus  of  malignant 
oedema,  and  the  bacillus  of  symptomatic  anthrax. 


CULTIVATION   OF    ANAEROBIC   BACTERIA.  81 

If  we  make  an  inoculation  of  one  of  the  species  which  is  not 
strictly  anaerobic  into  a  test  tube  containing  nutrient  gelatin  or  agar- 
agar,  we  may  have  a  development  all  along  the  line  of  puncture, 
and  this  may  be  more  abundant  below,  as  in  Fig.  47.  But  when  we 
make  a  long  stab  culture  with  a  strict  anaerobic  the  development 
occurs  only  near  the  bottom  of  the  line  of  puncture  (Fig.  48). 

We  may  then,  if  we  have  a  pure  culture  to  start  with,  propagate 
these  anaerobic  bacilli  in  long  stab  cultures.  It  is  best  to  use  tubes 
which  have  been  recently  sterilized,  as  boiling  expels  the  air  from 
the  culture  medium ;  and  a  very  slender  needle  should  be  used  in 
making  the  inoculation.  To  prevent  the  absorption  of  oxygen  a 
layer  of  sterilized  olive  oil  may  be  poured  into  the  tube  after  the  in- 
oculating puncture  has  been  made,  or  it  may  be  filled  up  with  agar 
jelly  which  has  been  cooled  to  about  40°  C.  Roux  has  proposed  to 
prevent  the  absorption  of  oxygen  by  the  culture  medium  by  plant- 
ing an  aerobic  bacterium— Bacillus  subtilis — upon  the  surface,  after 
making  a  long  stab  culture  with  the  anaerobic  species.  The  agar 
jelly  is  first  boiled  and  quickly  cooled  ;  the  inoculation  is  then  made 
with  a  slender  glass  needle  ;  some  sterile  agar  cooled  to  40°  C.  is 
poured  into  the  tube,  and  when  this  is  solid  the  aerobic  species  is 
planted  upon  the  surface.  The  top  of  the  test  tube  is  then  closed 
hermetically  and  it  is  placed  in  the  incubating  oven.  The  aerobic 
species  exhausts  the  oxygen  in  the  upper  part  of  the  tube  by  its 
growth  on  the  surface  of  the  culture  medium,  and  the  anaerobic 
species  grows  at  the  bottom  of  the  tube.  To  obtain  material  for  a 
new  culture  or  for  microscopical  examination  the  test  tube  is  broken 
near  its  bottom. 

Cultures  in  liquid  media  may  be  made  by  exhausting  the  air  in 
a  suitable  recaptacle  or  by  displacing  it  with  hydrogen  gas.  The 
first-mentioned  method  has  been  largely  used  in  Paste  :ir's  laboratory, 
but  methods  in  which  hydrogen  gas  takes  the  place  of  atmospheric 
air  in  the  culture  tube  are  more  easily  applied  an  -1  require  simpler 
apparatus.  The  flask  shown  in  Fig.  49  may  be  u  ;od  in  connection 
with  an  air  pump.  The  sterile  culture  liquid  is  firjt  introduced  into 
a  long-necked  flask  and  inoculated  with  the  anaerobic  bacillus  to  be 
cultivated.  The  neck  of  the  flask  is  then  drawn  out  in  a  flame  at  c. 
The  open  end  is  then  connected  with  a  Sprengle's  pump  or  some 
other  apparatus  for  exhausting  the  air.  The  flask  is  placed  in  a 
water  bath  at  40°  C.,  which  causes  ebullition  at  the  diminished  pres- 
sure, and  the  exhaustion  is  continued  for  about  half  an  hour.  The 
narrow  neck  is  then  sealed  at  c  by  the  use  of  a  blowpipe  flame. 

The  flask  shown  in  Fig.  49,  which  can  be  made  from  a  test  tube, 
may  also  be  used  in  connection  with  a  hydrogen  apparatus.     In  this 
case  a  slender  glass  tube  is  passed  into  the  flask,  as  shown  in  Fig. 
6 


82  CULTIVATION   OF    ANAEROBIC   BACTERIA. 

50,  and  this  is  connected  with  a  hydrogen  apparatus  by  a  rubber 
tube,  The  hydrogen  is  allowed  to  bubble  through  the  culture 
liquid  in  a  full  stream  for  ten  to  fifteen  minutes,  in  order  that  all  of 
the  oxygen  in  the  flask  may  be  removed  by  displacement.  Then, 
while  the  gas  is  still  flowing,  the  flask  is  sealed  at  a  with  a  blow- 
pipe flame,  the  hydrogen  tube  being  left  in  position  and  melted  fast 
to  the  flask.  Some  little  skill  is  required  in  the  successful  perform- 
ance of  the  last  step  in  this  procedure,  and  it  will  be  easier  for  those 


a 


\ 


FIQ.  49. 


FIG.  51. 


who  are  not  skilful  in  the  use  of  the  blowpipe  to  use  Salomonson's 
tube,  shown  in  Fig.  51.  In  this,  hydrogen  is  admitted  through  the 
arm  6,  and  escapes  through  the  cotton  plug  a.  The  vertical  tube  is 
sealed  at  c  while  the  gas  is  flowing,  and  then  the  horizontal  tube  at  b. 
Frankel's  Method. — Instead  of  these  tubes  specially  made  for 
the  purpose,  an  ordinary  test  tube  may  be  used,  as  recommended  by 
Frankel.  This  is  closed  by  a  soft  rubber  cork  through  which  two 
glass  tubes  pass — one,  reaching  nearly  to  the  bottom  of  the  test  tube, 


CULTIVATION   OF   ANAEROBIC    BACTERIA. 


83 


for  the  admission  of  hydrogen,  which  passes  through  the  liquefied 
culture  medium  ;  and  the  other  a  short  tube  for  the  escape  of  the  gas. 
The  outlet  tube  is  sealed  in  the  flame  of  a  lamp  while  the  gas  is 
freely  flowing,  and  after  sufficient  time  has  elapsed  to  insure  the 
complete  expulsion  of  atmospheric  oxygen — which,  when  the  hydro- 
gen flows  freely,  requires  about  four  minutes  (Frankel) — melted 
paraffin  is  applied  freely  to  the  rubber  stopper  to  prevent  leakage  of 
the  hydrogen  and  entrance  of  oxygen.  A  roll  tube  may  then  be 
made  after  the  manner  of  Esmarch,  and,  after  colonies  have  de- 
veloped, the  anaerobic  culture  will  appear  as  shown  in  Fig.  52. 

To  isolate  anaerobic  bacteria  in  pure  cultures  it  is  well  to  make  a 


-t 


-a 


\J 

FIG.  53. 

series  of  dilutions  as  heretofore  described  for  aerobic  cultures  ;  we 
will  then  usually  obtain  isolated  colonies  in  tube  No.  2  or  No.  3  of  a 
series,  and  by  removing  the  rubber  stopper  we  may  transplant  bac- 
teria from  these  colonies  to  deep  stab  cultures  in  nutrient  gelatin  or 
agar. 

The  Writer's  Method.— The  following  simple  method  has  been 
successfully  employed  by  the  writer: 

Three  Esmarch  roll  tubes  are  prepared  as  is  usual  for  aerobic  cul- 
tures. The  cotton  air  filter,  or  a  portion  of  it,  is  then  pushed  down 
the  tubes  for  a  short  distance,  as  shown  at  a,  Fig.  53.  A  section  of 
a  soft  rubber  stopper  carrying  two  glass  tubes  is  then  pushed  into  the 


84 


CULTIVATION    OF   ANAEROBIC    BACTERIA. 


test  tube  for  about  half  an  inch,  as  shown  at  b,  Fig.  53.  The  space 
above  the  cork  is  then  filled  with  melted  sealing  wax,  which  I  have 
found  to  prevent  leakage  better  than  paraffin,  which  contracts  upon 
cooling.  The  test  tube  is  inverted  while  hydrogen  is  passed  through 
the  tube  c,  and  by  reason  of  its  levity  the  gas  quickly  passes  through 
the  cotton  air  filter  and  displaces  the  oxygen  in  the  test  tube  (Fig. 
54).  After  allowing  the  gas  to  flow  for  a  few  minutes  the  outlet 
tube  is  first  sealed  in  a  flame  and  then  the  inlet  tube.  As  the  cotton 
filter  is  interposed  between  the  rubber  stopper  and  the  culture  mate- 
rial, no  special  precautions  need  be  taken  for  the  sterilization  of  the 
rubber  cork  and  the  glass  tubes  which  it  carries. 


FIG.  54. 


FIG.  55. 


This  method  is  more  convenient  than  that  previously  described, 
and  the  only  objection  to  it  is  that  the  oxygen  is  not  completely  re- 
moved from  the  film  of  solid  gelatin  or  agar  attached  to  the  walls  of 
the  test  tube.  But  by  passing  the  hydrogen  for  a  long  time  it  would 
seem  that  by  diffusion  the  oxygen  remaining  in  this  thin  layer 
would  be  gotten  rid  of.  At  all  events,  this  method  will  serve  for  all 
except  the  very  strict  anaerobics. 

Method  of  Es march.—  The  following  method  has  been  proposed 
by  Esmarch  :  Three  roll  tubes  are  made  in  the  usual  way,  and  into 
these  liquid  gelatin,  that  is  nearly  cooled  to  the  point  of  becoming 
solid,  is  poured.  This  fills  the  tube  without  melting  the  layer  of 


CULTIVATION   OF    ANAEROBIC    BACTERIA. 


85 


gelatin,  previously  cooled  upon  its  walls,  which  contains  the  bacteria 
under  investigation.  When  the  anaerobic  colonies  have  developed 
the  test  tube  must  be  broken  to  get  at  them,  or  the  cylinder  of  gela- 
tin may  be  removed  by  first  warming  the  walls  of  the  tube. 

Another  method,  recommended  by  Liborius,  consists  in  distri- 
buting the  bacteria  in  test  tubes  nearly  filled  with  nutrient  gelatin  or 
agar  which  has  been  recently  boiled  to  expel  air.  Colonies  of  anaero- 
bic bacteria  will  develop  near  the  bottom  of  such  a  tube,  while  the 
aerobic  species  will  only  grow  near  the  surface.  The  cylinder  of 
jelly  is  removed  by  heating  the  walls  of  the  tube,  and  sections  are 


fr  a 


Fia.  56. 

made  with  a  sterilized  knife  for  the  purpose  of  'obtaining  material 
from  individual  colonies  for  further  cultures,  etc. 

Koch  and  his  pupils  are  in  the  habit  of  testing  the  aerobic  char- 
acter of  bacteria  in  plate  cultures  by  covering  the  recently  made 
plates  with  a  thin  sheet  of  mica  which  has  been  sterilized  by  heat. 
The  strictly  aerobic  species  do  not  grow  under  such  a  plate  ;  but, 
according  to  Liborius,  the  exclusion  of  oxygen  is  not  sufficiently 
complete  for  the  growth  of  strict  anaerobics. 

Buclmer's  Method  consists  in  the  removal  of  oxygen  by  means 
of  pyrogallic  acid.  The  anaerobic  species  under  investigation  is 
planted  in  recently  boiled  agar  jelly  in  a  small  test  tube.  This  is 
placed  in  a  larger  tube  having  a  tightly  fitting  rubber  stopper,  as 
shown  in  Fig.  55.  The  small  tube  is  supported  by  a  bent-wire 


8G 


CULTIVATION   OF  ANAEROBIC   BACTERIA. 


stand,  and  in  the  lower  part  of  the  large  tube  are  placed  ten  cubic 
centimetres  of  a  ten-per-ceiit  solution  of  caustic  potash,  to  which  one 
gramme  of  pyrogallic  acid  is  added.  The  absorption  of  the  oxygen 
takes  some  time,  but,  according  to  Buchner,  it  is  finally  so  complete 
that  strict  anaerobics  grow  in  the  small  tube. 

In  practice,  cultivation  in  an  atmosphere  of  hydrogen  will  be 
found  the  most  convenient  method,  and  for  this  any  form  of  hydro- 
gen generator  may  be  used.  The  writer  is  in  the  habit  of  using  the 
form  shown  in  Fig.  56.  A  perforation  a  quarter  of  an  inch  in 
diameter  is  drilled  through  the  bottom  of  a  wide-mouthed  bottle. 
Some  fragments  of  broken  glass  are  then  put  into  the  bottle,  form- 


FIG.  57. 

ing  a  layer  two  or  three  inches  thick.  Upon  this  is  placed  a  quan- 
tity of  granulated  zinc.  This  bottle  has  a  tightly  fitting  cork, 
through  which  passes  a  metal  tube  having  a  stopcock.  The  bottle 
is  placed  in  a  glass  jar  containing  diluted  sulphuric  acid  (one  part 
by  weight  of  sulphuric  acid  to  eight  parts  of  water).  The  acid,  ris- 
ing through  the  perforation  in  the  bottom  of  the  bottle,  when  it 
comes  in  contact  with  the  zinc  gives  rise  to  an  abundant  evolution 
of  hydrogen,  which  escapes  by  the  tube  a  when  the  stopcock  is 
open.  When  this  is  closed  the  gas  forces  the  acid  back  from  con- 
tact with  the  zinc.  To  remove  any  trace  of  oxygen  present  the 
gas  may  be  passed  through  a  solution  of  pyrogallic  acid  in  caustic 
potash. 

Evidently  plates  prepared   by  Koch's  method,  or  Esmarch  roll 


CULTIVATION   OF   ANAEROBIC   BACTERIA.  87 

tubes,  may  be  placed  in  a  suitable  receiver  and  the  air  exhausted,  or 
hydrogen  substituted  for  atmospheric  air.  Such  an  apparatus  for 
hydrogen  has  been  devised  by  Bliicher  and  is  shown  in  Fig.  57.  A 
glass  dish,  A,  contains  a  smaller  dish,  B,  which  has  a  diameter  of 
about  seven  centimetres.  The  small  dish  is  kept  in  its  position  in 
the  centre  of  the  larger  one  by  the  wire  ring,  having  three  project- 
ing arms,  which  is  shown  in  the  figure.  The  culture  medium  con- 
taining the  anaerobic  bacteria  to  be  cultivated  is  poured  into  the 
small  dish  and  the  glass  funnel  D  is  put  in  position.  This  is  held 
in  its  place  by  a  weight  of  lead  which  encircles  the  neck  of  the  fun- 
nel at  F.  A  mixture  of  glycerin  and  water  (twenty  to  twenty-five 
per  cent)  is  poured  into  the  dish  A  to  serve  as  a  valve  to  shut  off 
the  atmospheric  air  from  the  interior  of  the  funnel  D.  Hydrogen 
gas  is  introduced  through  the  tube  E,  which  is  connected  by  a  rub- 
ber tube  with  a  hydrogen  apparatus. 

A  somewhat  similar  apparatus  has  been  devised  by  Botkin,  in 
which  the  hydrogen  is  admitted  beneath  a  bell  jar  covering  small 
glass  dishes  containing  the  culture  medium.  We  believe  that  in 
practice  the  writer's  method  (page  83),  in  which  Esmarch  roll  tubes 
are  first  made,  will  be  found  more  convenient  than  either  of  the  last- 
mentioned  methods  of  preserving  plates  in  an  atmosphere  of  hydro- 
gen ;  or  roll  tubes  may  be  prepared  in  the  way  usually  practised  in 
cultivating  aerobic  bacteria,  and  these  may  be  placed  in  a  suitable 
receptacle  which  can  be  filled  with  hydrogen. 

The  addition  of  a  reducing  agent  to  the  culture  medium  favors 
the  growth  of  anaerobic  bacteria.  Kitasato  and  Weil  have  recom- 
mended formic  acid  or  sodium  formate,  in  the  proportion  of  0.3  to  0.5 
per  cent.  Theobald  Smith  has  found  0.3  to  0.5  per  cent  of  glucose 
to  be  a  useful  addition  with  the  same  object  in  view. 


X. 
INCUBATING   OVENS   AND   THERMO-REGULATORS. 

THE  saprophytic  bacteria  generally,  and  many  of  the  pathogenic 
species,  grow  at  the  ordinary  temperature  of  occupied  apartments 
(20°  to  25°  C. )  ;  but  some  pathogenic  species  can  only  be  cultivated 
at  a  higher  temperature,  and  many  of  those  which  grow  at  the 
"room  temperature"  develop  more  rapidly  and  vigorously  when 
kept  in  an  incubating  oven  at  a  temperature  of  35°  to  38°  C.  Every 
bacteriological  laboratory  should  therefore  be  provided  with  one  or 
more  brood  ovens  provided  with  thermo-regulators  to  maintain  a 
constant  temperature.  These  incubating  ovens  are  made  with  dou- 
ble walls  surrounding  an  air  chamber.  The  space  between  the  dou- 
ble walls  is  filled  with  water,  which  is  usually  heated  by  a  small  gas 
flame.  The  gas  passes  through  the  thermo-regulator,  and  its  flow 
is  automatically  controlled  for  any  temperature  to  which  this  is  ad- 
justed. The  exterior  of  the  incubating  oven  is  covered  with  felt  or 
asbestos  to  prevent  the  loss  of  heat  by  radiation.  A  simple  and 
cheap  form  which  answers  every  purpose  is  shown  in  Fig.  58.  The 
quadrangular  box  with  double  walls  should  be  made  of  zinc  or  cop- 
per. An  outer  metal  door  covered  with  non-conducting  material, 
and  an  inner  door  of  glass,  give  access  to  the  interior  space  ;  and  a 
thermometer  introduced  through  an  aperture  in  the  top  (Fig.  58,  b) 
shows  the  temperature  of  this  space  when  the  door  is  closed.  The 
stopcock  e  permits  the  drawing  off  of  the  water  from  the  space  be- 
tween the  double  walls,  and  the  glass  tube  d  shows  the  height  of 
the  water,  as  it  is  connected  with  the  space  containing  it.  The 
thermo-regulator  passes  through  an  aperture  at  one  side  of  the  oven 
into  the  water,  the  temperature  of  which,  controls  the  flow  of  gas. 

The  ordinary  thermo-regulator  is  shown  in  Fig.  50  as  manufac- 
tured by  Rohrbeck.  A  glass  receptacle,  shaped  like  an  ordinary 
test  tube,  has  an  arm,  c,  for  the  escape  of  the  gas,  which  enters  by 
the  bent  tube  a,  which  passes  through  a  perforated  cork  and  is  ad- 
justable up  and  down.  Tube  a  is  connected  with  the  gas  supply  and 
tube  c  with  the  burner  by  means  of  rubber  tubing.  A  glass  parti- 
tion extending  downward  as  a  tube,  g,  makes  an  enclosed  space  in 


INCUBATING   OVENS   AND    THERMO-REGULATORS. 


89 


the  lower  part  of  the  instrument,  and  this,  when  immersed  in  water, 
acts  as  a  thermometer  bulb.  This  space  contains  mercury  below 
and  air  or  the  vapor  of  ether  above.  When  the  air  is  expanded  by 
heat  the  mercury  is  forced  up  the  tube  g  until  it  meets  the  end  of 
the  inlet  tube  for  gas  at  h,  and  by  shutting  off  the  flow  of  gas  pre- 
vents the  temperature  from  going  any  higher.  A  small  opening  in 
the  inlet  tube  at  e  permits  a  small  amount  of  gas  to  flow,  so  that  the 
flame  under  the  brood  oven  ( Fig.  58,  /)  may  not  be  entirely  extin- 
guished. The  lower  end  of  the  bout  tube  a  is  bevelled,  so  that  a  tri- 
angular opening  is  formed,  which  is  closed  gradually  by  the  rising 


FIG.  58. 

mercury,  instead  of  abruptly  as  would  be  the  case  if  the  lower  end 
of  the  tube  a  were  cut  off  square.  To  adjust  the  temperature  in 
the  air  space  of  the  incubating  oven  when  the  thermo-regulator  is  in 
position,  a  full  flow  of  gas  is  admitted  to  the  burner  until  the  ther- 
mometer (Fig.  58,  b)  shows  the  desired  temperature ;  then  the  bent 
tube  a  is  pushed  down  through  the  cork  until  its  lower  extremity 
meets  the  mercury  and  the  flame  /  is  somewhat  reduced.  The  ap- 
paratus is  then  left  for  a  time,  to  see  whether  the  flame  runs  too  high 
or  too  low,  and  a  further  adjustment  is  made.  When  the  changes 
in  the  exterior  temperature  are  slight  and  the  gas  pressure  regular 
the  temperature  in  the  air  chamber  is  controlled  with  great  precision. 
But  this  is  not  the  case  under  the  reverse  conditions.  Changes  in 


90 


INCUBATING   OVENS   AND    THERMO-REGULATORS. 


the  pressure  of  gas,  especially,  interfere  with  the  maintenance  of  a 
constant  temperature,  and  for  this  reason  a  pressure  regulator  will 
be  required  when  great  precision  is  desired.  That  of  Moitessier  is 
commonly  used  in  bacteriological  laboratories  (Fig.  60).  But  for 
most  purposes  variations  of  temperature  of  1°  to  2°  C.  are  not  of 
great  importance.  For  ordinary  use  a  brood  oven  should  be  regu- 
lated to  about  35°  to  37°  C.  It  is  best  to  have  a  little  cylindrical 
screen  of  mica  around  the  gas  jet  beneath  the  incubating  oven,  for 
the  purpose  of  preventing  the  flame  from  being  extinguished  by  cur- 
rents of  air  (Fig.  61). 

Koch's  ingenious  automatic  device  for  shutting  off  the  gas  if  the 
flame  is  accidentally  extinguished  is  shown  in  Fig.  62. 


a 


FIG.  59. 


FIG.  60. 


Another  form  of  thermo-regulator,  which  answers  very  well,  is 
that  of  Reichert  (Fig.  63).  In  this  the  gas  enters  at  a  and  escapes 
at  c.  The  mercury,  which  fills  the  bulb,  shuts  off  the  gas  at  the 
point  for  which  the  instrument  is  regulated.  By  means  of  the 
screw  d  the  height  of  the  mercury  in  the  tube  may  be  very  accu- 
rately adjusted  for  any  desired  temperature. 

The  regulator  of  Bohr,  shown  in  Fig.  64,  is  more  sensitive  than 
that  of  Reichert,  and  rather  simpler  in  construction  than  the  usual 
form  shown  in  Fig.  59.  The  thermometer  bulb  a  contains  only  air, 
and  the  gas  which  passes  through  the  tube  /  is  shut  off  at  the 
proper  temperature  by  the  mercury  in  the  U-shaped  tube  c.  The 
stppcock  b  is  left  open  when  the  bulb  a  is  immersed  in  the  water 


INCUBATING    OVENS   AND    THERMO-REGULATORS. 


91 


bath,  and  when  the  proper  temperature  is  reached  is  closed  so  as  to 
confine  the  air  in  the  bulb.     An  increase  of  temperature  now  causes 


FIG.  61.  FIG.  62. 

the  air  in  the  bulb  to  expand,  the  mercury  in  the  U-tube  is  forced  up 


...a 


FIG.  63. 


FIG.  64. 


and  shuts  off  the  gas  flowing  through  the  tube /at  its  lower  ex- 
tremity,  d.     A  small  opening,  e,  permits  sufficient  gas  to  pass  to 


92  INCUBATING   OVENS   AND    THERMO-REGULATORS. 

maintain  a  small  flame  which  must  not  be  sufficient  by  itself  to  keep 
up  the  desired  temperature  in  the  water  bath. 

Altmann  has  recently  (1891)  described  a  thermo-regulator  which 
is  made  by  Miincke,  of  Berlin,  and  which  is  shown  in  Fig.  65.  This 
is  said  to  act  with  great  precision.  It  is  a  modification  of  Reichert's 


FIG.  65. 


FIG.  <>6. 


regulator.     Its  mode  of  action  will  be  readily  understood  by  a  refe- 
rence to  the  figure. 

A  thermo-regulator  which  gives  very  accurate  results,  which  are 
not  influenced  by  differences  in  pressure,  is  that  invented  by  the 


FIG.  67. 

writer  over  thirty  years  ago.  The  regulating  thermometer  may 
contain  mercury  only,  or  air  and  mercury,  as  shown  in  the  thermo- 
regulator  for  gas  (Fig.  59).  In  the  simplest  form  a  large  bulb  con- 
taining mercury  is  used,  and  a  platinum  wire  is  hermetically  sealed 
in  the  glass  so  as  to  have  contact  with  the  mercury  (Fig.  66,  a). 


INCUBATING    OVENS   AND    THERMO-REGULATORS.  93 

Another  platinum  wire  passes  down  the  tube  of  the  thermometer,  b, 
and  is  adjustable  for  any  desired  temperature.  The  gas  passes 
through  a  valve  which  is  controlled  by  an  electro-magnet.  A 
simple  form  of  valve  is  shown  in  Fig.  G7.  The  bent  tube  a  is  con- 
nected with  the  gas  supply  by  a  piece  of  rubber  tubing.  The  up- 
right arm  of  this  tube  is  enclosed  in  a  larger  tube,  b,  having  an  out- 


FIG.  68.  FIG.  69. 

let,  e,  which  is  connected  with  the  burner  under  the  incubating 
oven.  The  upper  end  of  this  larger  tube  is  closed  by  means  of  a 
piece  of  sheet  rubber,  which  prevents  the  escape  of  gas.  When  this 
is  depressed  by  means  of  the  lever  c,  the  flow  of  gas  through  the 
valve  is  arrested.  The  lever  c  has  attached  to  it  the  armature  d, 
and  is  operated  by  an  electro-magnet  under  the  control  of  the  regu- 
lating thermometer. 


94 


INCUBATING   OVENS   AND    THERMO-REGULATORS. 


"When  the  thermometer  is  immersed  in  a  water  bath  the  tem- 
perature of  which  it  is  desired  to  regulate,  and  the  proper  electric 
connections  are  made,  it  acts  as  a  circuit  breaker.  When  the  de- 
sired temperature  is  reached  the  mercury  in  the  tube  of  the  ther- 
mometer touches  the  wire  b  (Fig.  66),  an  electric  circuit  is  com- 
pleted, and  the  valve  is  closed,  shutting  off  the  gas  supply  and 
preventing  the  temperature  from  going  any  higher.  When  contact 
is  broken  in  the  thermometer  tube  the  valve  opens  and  permits  the 
gas  to  flow  again.  A  small  opening,  o  (Fig.  67),  permits  the  con- 
stant flow  of  a  sufficient  amount  of  gas  to  prevent  the  flame  from 
being  extinguished.  In  practice,  however,  it  is  better  to  have  a 
small  side  jet  of  gas,  quite  independent  of  that  which  passes  through 
the  valve,  which  burns  constantly  and  relights  the  principal  jet  when 


FIG.  70. 

the  valve  is  opened.  This  apparatus  is  very  well  adapted  for  regu- 
lating the  temperature  of  a  water  bath  with  precision,  but  for  gene- 
ral use  in  connection  with  incubating  ovens  the  ordinary  gas  regu- 
lator is  preferable,  on  account  of  the  trouble  connected  with  keeping 
a  galvanic  battery  in  order  when  it  is  required  to  act  at  frequent 
intervals  "  on  a  closed  circuit/'  for  weeks  and  months  together. 

The  incubating  apparatus  of  D'Arsoiival  is  shown  in  Fig.  68.  It 
is  a  cylindrical  vessel  of  copper  having  double  walls,  and  is  provided 
with  the  thermo-regulator  of  D7  Arson  val,  by  which  very  accurate 
regulation  is  maintained  at  any  desired  temperature.  In  its  form 
this  apparatus  is  not  as  convenient  as  are  the  brood  ovens  made 
in  the  form  shown  in  Fig.  58,  with  a  swinging  door  which  gives 
easy  access  to  the  interior,  which  is  provided  with  one  or  more 
shelves  upon  which  the  cultures  are  placed.  Various  modifications 


INCUBATING    OVENS   AND    THERMO-REGULATORS.  95 

of  this  simple  and  convenient  incubating-  oven  are  manufactured 
by  Robrbeck  and  by  Muncke,  of  Berlin.  The  apparatus  of  D'Ar- 
sonval,  and  other  forms  in  favor  at  the  French  capital,  may  be  ob- 
tained from  Wiesnegg,  of  Paris.  The  last-named  manufacturer 
also  supplies  the  incubating  oven  and  thermo-regulator  described  by 
Roux  (1891).  This  is  shown  in  Fig.  69.  The  regulator  is  formed  of 
two  metallic  bars,  one  of  steel  and  the  other  of  zinc  ;  these  are 
soldered  together  in  the  shape  of  a  letter  (J  ;  the  regulator  is  seen  in 
position  in  the  cut  (Fig.  G9).  The  most  dilatable  metal  (zinc)  is  on 
the  outside.  When  the  temperature  is  raised  the  arms  of  the  (J  ap- 
proach each  other,  and  the  reverse  when  it  falls.  The  method  by 
which  regulation  is  effected  is  shown  in  Fig.  70.  The  U-shaped 
regulator  is  placed  vertically,  and  one  of  its  branches,  A,  is  firmly 
fixed  to  the  wall  of  the  incubating  oven  ;  the  other,  free  arm  car- 
ries a  horizontal  bar  which  projects  through  the  wall  of  the  incu- 
bator in  an  opening  which  permits  it  to  move  freely  under  the  influ- 
ence of  a  change  in  the  temperature  within.  The  end  of  this 
projecting  bar  is  turned  up  at  a  right  angle  and  the  screw  p  passes 
through  it ;  this  can  be  fixed  at  any  desired  point  by  means  of  the 
nut  e.  The  end  of  the  screw  p  rests  against  the  stem  of  a  conical 
brass  valve  which  controls  the  flow  of  gas.  The  valve  is  closed  by  a 
spiral  spring  and  opened  by  the  screw  p  under  the  control  of  the 
thermo-regulator. 

In  the  absence  of  gas  incubating  ovens  may  be  heated  by  a  small 
petroleum  lamp,  and  various  devices  have  been  invented  for  control- 
ling the  temperature.  Reichenbach  describes  an  apparatus  for  this 
purpose  in  the  Centralblatt  fur  Bakteriologie,  Vol.  XV.,  p.  847, 
1894.  Dr.  Borden  of  the  U.  S.  Army  has  also  invented  a  thermo- 
regalator  to  be  used  in  connection  with  a  petroleum  lamp.  In  the 
absence  of  any  regulating  apparatus  an  incubating  oven  may  be  kept 
at  a  tolerably  uniform  temperature  by  personal  supervision — adjusting 
the  flame  of  the  lamp  and  its  distance  from  the  bottom  of  the  oven  ac- 
cording to  the  changes  in  the  external  temperature.  For  most  bac- 
teria a  variation  of  several  degrees  is  not  important,  so  long  as  the 
temperature  is  not  allowed  to  rise  above  37°  to  38°  C.  The  typhoid 
bacillus,  the  diphtheria  bacillus,  the  anthrax  bacillus,  the  pus  cocci, 
and  most  saprophytic  bacteria  grow  at  the  ordinary  room  temperature, 
ap<l  may  therefore  be  cultivated  without  any  form  of  incubating  oven 
01  thermo-regulator. 


XI 

EXPERIMENTS   UPON   ANIMALS. 

THE  pathogenic  power  of  various  bacteria  has  been  demonstrated 
by  injecting  pure  cultures  into  susceptible  animals.  As  a  rule,  the 
herbivora  are  more  susceptible  than  the  carnivora,  and  this  is  per- 
haps to  be  explained  in  accordance  with  the  theory  of  natural  selec- 
tion. Carnivorous  animals  often  feed  upon  the  bodies  of  animals 
which  have  succumbed  to  infectious  diseases,  and  upon  dead  animals 
in  which  putrefactive  changes  have  commenced.  In  their  struggles 
with  each  other  they  are  wounded  by  teeth  and  claws  soiled  with  in- 
fectious material  which  would  cause  a  fatal  disease  if  inoculated  into 
the  more  susceptible  herbivorous  animals.  As  this  has  been  going 
on  for  ages,  we  may  suppose  that,  by  survival  of  the  fittest,  a  race 
tolerance  has  been  acquired.  The  lower  animals  have  their  own  in- 
fectious diseases,  some  of  which  are  peculiar  to  certain  species  and 
some  common  to  several.  As  a  rule,  the  specific  infectious  diseases 
of  man  cannot  be  transmitted  to  lower  animals,  and  man  is  not  sub- 
ject to  the  diseases  of  the  same  class  which  prevail  among  animals. 
But  certain  diseases  furnish  an  exception  to  this  general  rule.  Thus 
tuberculosis  is  common  to  man  and  several  of  the  lower  animals ; 
relapsing  fever  may  by  inoculation  be  transmitted  to  monkeys  ; 
diphtheria  may  be  transmitted  to  pigeons  and  guinea-pigs.  On  the 
other  hand,  anthrax  and  glanders  may  be  contracted  by  man  as  a 
result  of  accidental  inoculation  or  contact  with  an  infected  animal. 

Nearly  allied  species  sometimes  present  very  remarkable  differ- 
ences as  to  susceptibility.  Thus  the  bacillus  of  mouse  septicaemia  is 
fatal  to  house  mice  but  not  to  field  mice,  while,  on  the  other  hand, 
field  mice  are  killed  by  the  bacillus  of  glanders  and  house  mice  are 
immune  from  this  pathogenic  bacillus. 

The  animals  most  commonly  used  for  testing  the  pathogenic 
power  of  bacteria  are  the  mouse,  the  guinea-pig,  and  the  rabbit. 
Domestic  fowls  and  pigeons  are  also  useful  for  certain  experiments. 
The  dog  and  the  rat  are  of  comparatively  little  use  on  account  of 
their  slight  susceptibility. 


EXPERIMENTS   UPON   ANIMALS. 


97 


Inoculations  are  made  directly  into  the  circulation  through  a 
vein,  into  the  subcutaneous  connective  tissue,  or  into  one  of  the 
serous  cavities — usually  the  peritoneal. 

The  ordinary  hypodermic  syringe  may  be  used  in  making  injec- 
tions, but  this  is  difficult  to  sterilize  on  account  of  the  leather  piston, 
and  complications  are  liable  to  arise  from  its  use  which  it  is  best  to 
avoid.  The  best  way  to  sterilize  a  piston  syringe  is  to  wash  it  thor- 
oughly with  a  solution  of  bichloride  of  mercury  of  1  : 1,000,  and  then 
to  remove  every  trace  of  bichloride  by  washing  in  alcohol.  But  one 
never  feels  quite  sure  that  the  most  careful  washing  will  insure  steril- 
ization, and  it  is  best  to  use  a  syringe  which  may  be  sterilized  by 


FIG  71. 


heat,  such  as  that  of  Koch,  shown  in  Fig.  71.  In  this  the  metal  point 
and  glass  tube  are  easily  sterilized  in  a  hot-air  oven.  Fluid  is  drawn 
into  the  syringe  and  forced  out  of  it  by  a  rubber  ball  which  has  a 
perforation  to  be  covered  by  the  finger. 

The  writer  has  for  some  years  boen  in  the  habit  of  making  injec- 
tions in  animals  with  an  improvised  glass  syringe.  This  is  made 
from  a  piece  of  glass  tubing  in  the  same  form  as  the  collecting  tubes 
heretofore  described.  A  bulb  is  blown  at  one  end  of  the  tube,  and 
the  other  end  is  drawn  out  to  form  a  slender  tube  which  serves  as  the 


FIG.  72. 

needle  of  the  syringe  (Fig.  72).  By  gently  heating  the  bulb  in  an 
alcohol  lamp  and  immersing  the  open  end  of  the  capillary  tube  in 
the  fluid  to  be  injected,  this  rises  into  the  syringe  as  the  expanded  air 
cools.  Having  introduced  the  glass  point  beneath  the  skin  or  into 
the  cavity  of  the  abdomen  of  the  animal  to  be  injected,  the  contents 
of  the  tube  are  forced  out  by  again  heating  the  bulb  by  means  of  a 
small  alcohol  lamp.  The  glass  point  is  easily  forced  through  the 
thin  skin  of  a  mouse  or  of  a  young.rabbit ;  but  for  animals  with  a 
thicker  skin  it  is  necessary  to  cut  through,  or  nearly  through,  the 
skin  with  some  other  instrument.  A  small  pair  of  curved  scissors, 
answers  very  well  for  this  purpose. 


98  EXPERIMENTS   UPON   ANIMALS. 

Generally,  in  making  injections  into  animals,  it  is  customary  to 
remove  the  hair  for  some  distance  around  the  point  of  inoculation 
with  scissors  and  razor,  and  then  to  sterilize  the  surface  by  careful 
washing  with  a  solution  of  bichloride  of  mercury.  This  precaution 
is  necessary  in  researches  in  which  pathogenic  bacteria  are  being 
tested,  in  order  to  remove  any  possibility  of  accidental  inoculation 
with  germs  other  than  those  under  investigation,  and,  as  a  conse- 
quence, a  mistaken  inference  as  to  the  pathogenic  action  of  the  spe- 
cies under  investigation.  But  when  we  know  the  specific  pathogenic 
power  of  a  certain  microorganism  it  is  hardly  necessary  to  take  this 
precaution,  as  a  few  drops  of  culture  will  contain  millions  of  the  bac- 
teria, while  contamination,  if  it  occurs  from  the  surface  of  the  body, 
must  be  by  a  comparatively  small  number  of  bacteria,  which  are 
likely  to  be  of  a  harmless  kind  which  will  have  no  influence  on  the 
result  of  the  experiment. 

Instead  of  sterilizing  the  surface,  the  writer  usually  clips  away  a 
small  portion  of  skin  with  curved  scissors,  not  cutting  deep  enough 
to  draw  blood,  but  leaving  a  bare  surface  through  which  the  point  of 
the  syringe  can  be  introduced  with  very  little  danger  of  carrying  bac- 
teria into  the  connective  tissue  other  than  those  contained  in  the 
syringe.  * 

In  making  injections  into  the  peritoneal  cavity  care  must  be  taken 
not  to  wound  the  liver  or  the  distended  stomach.  The  intestine  is 
not  very  likely  to  be  wounded,  as  it  slips  out  of  the  way.  By  seizing 
a  longitudinal  fold  of  the  abdominal  wall  and  pushing  the  point  of 
the  syringe  quite  through  it,  and  then  releasing  the  fold  and  care- 
fully withdrawing  the  instrument  until  the  point  remains  in  the 
cavity,  the  danger  of  wounding  the  intestine  will  be  reduced  to  a 
minimum. 

Injections  into  the  circulation  are  made  by  exposing  a  vein  and 
carefully  introducing  the  needle  of  the  syringe  in  the  direction  of 
the  blood  current.  Care  must  of  course  be  taken  not  to  inject  air. 
In  the  rabbit  one  of  the  large  veins  of  the  ear  may  be  conveniently 
penetrated  by  the  point  of  a  hypodermic  syringe  without  any  pre- 
vious dissection.  The  ear  is  first  washed  with  a  solution  of  bichloride 
of  mercury  or  simply  with  warm  water.  The  animal  had  better  be 
carefully  wrapped  in  a  towel  to  control  its  movements.  The  veins 
are  distended  by  compressing  them  near  the  base  of  the  ear.  When 
the  point  of  the  needle  has  not  been  properly  introduced,  and  the 
fluid  to  be  injected  escapes  in  the  surrounding  connective  tissue,  it 
will  commonly  be  best  to  withdraw  the  syringe  and  make  the 
attempt  upon  another  vein.  As  pointed  out  by  Abbott,  the  needle 
of  the  syringe  should  be  ground  flat  at  the  point,  and  not  curved  as 
is  commonly  the  case. 


EXPERIMENTS    UPON   ANIMALS.  99 

Large  quantities  of  fluid  may  be  injected  into  the  cavity  of  the 
abdomen  or  into  the  circulation  by  slowly  forcing  the  fluid  through 
a  slender  canula,  properly  introduced,  which  is  coupled  with  a  large 
syringe  by  means  of  rubber  tubing,  or  with  a  glass  receptacle  from 
which  the  fluid  is  forced  by  the  pressure  of  air  pumped  in  with  a 
rubber  hand  ball. 

Mice  are  usually  injected  subcutaneously  near  the  tail.  The 
little  animal  is  first  seized  by  a  long  pair  of  forceps,  or  "mouse 
tongs,"  and  the  hair  is  clipped  away  on  the  back  just  above  the  tail. 
If  solid  material  is  to  be  introduced  a  little  pocket  is  made  with  scis- 
sors or  with  a  lancet,  into  which  the  infectious  material  is  carried  by 
means  of  a  platinum  needle  or  slender  forceps.  Liquids  may  be  in- 
jected by  the  little  glass  syringe  heretofore  described,  the  point  of 
which  is  easily  forced  through  the  skin. 

Pasteur's  method  of  inoculating  rabbits  with  the  virus  of  hydro- 
phobia consists  in  trephining  the  skull  and  injecting  the  material 
beneath  the  dura  mater.  An  incision  through  the  skin  is  first  made 
to  one  side  of  the  median  line  a  short  distance  back  of  the  eyes. 
The  edges  of  the  wound  are  separated,  and  a  small  trephine  (five  or 
six  millimetres  in  diameter)  is  used  to  remove  a  button  of  bone.  The 
emulsion  of  spinal  cord  from  a  hydrophobic  animal  is  then  carefully 
injected  beneath  the  dura  mater — two  or  three  drops  will  be  sufficient. 
The  wound  is  washed  out  with  a  two-per-cent  solution  of  carbolic 
acid  and  closed  with  a  couple  of  sutures. 

Injections  into  the  intestine  are  made  by  carefully  opening  the 
abdomen  with  antiseptic  precautions,  gently  seizing  a  loop  of  the  in- 
testine, and  passing  the  point  of  the  syringe  through  its  walls  ;  the 
loop  is  then  returned  and  the  incision  in  the  walls  of  the  abdomen 
carefully  closed  with  sutures  and  dressed  antiseptically. 

Inoculations  into  the  anterior  chamber  of  the  eye  of  rabbits  and 
other  animals  have  frequently  been  practised,  and  offer  certain  ad- 
vantages in  the  study  of  the  local  effects  of  pathogenic  microorgan- 
isms. The  animal  should  be  fastened  to  an  operating  board,  belly 
down,  and  its  head  held  by  an  assistant,  who  at  the  same  time  holds 
the  eyelids  apart.  The  conjunctiva  is  seized  with  forceps  to  steady 
the  eye,  and  an  incision  about  two  millimetres  long  is  made  through 
the  cornea  with  a  cataract  knife.  Through  this  opening  a  small 
quantity  of  a  liquid  culture  may  be  injected,  or  a  bit  of  solid  material 
introduced  with  slender  curved  forceps. 

Ordinary  injections  give  but  little  pain  and  do  not  call  for  the  use 
of  an  anaesthetic.  When  anaesthesia  is  required  ether  will  usually 
be  preferable  to  chloroform.  Rabbits,  especially,  are  very  apt  to  die 
from  chloroform,  no  matter  how  carefully  it  may  be  administered. 
Dogs,  rats,  and  mice  stand  ether  very  well.  The  smaller  animals 


100  EXPERIMENTS    UPON   ANIMALS. 

may  be  brought  under  the  anaesthetic  by  placing  them  in  a  covered 
jar  into  which  a  pledget  of  cotton  wet  with  ether  has  been  dropped. 
Before  making  injections  into  the  anterior  chamber  of  the  eye  it  is 
well  to  use  a  two-per-cent  solution  of  cocaine  as  a  local  anesthetic. 

Mice  which  have  been  inoculated  are  usually  kept  in  a  glass  jar 
having  a  wire-gauze  cover.  A  quantity  of  cotton  is  put  into  the  jar 
to  serve  as  a  shelter  for  th#  little  animal,  and  it  is  well  to  partly  fill 
the  jar  with  dry  sawdust.  Larger  animals  are  kept  in  suitable  cages 
of  wire  or  wood,  and,  as  a  rule,  each  one  should  be  kept  in  a  separate 
cage  while  under  observation  after  an  inoculation  experiment. 

In  experimenting  upon  animals  the  following  points  should  be 
kept  in  view  and  noted  : 

(a)  The  age  and  weight  of  the  animal.     Young  animals  are,  as 
a  rule,  more  susceptible  than  older  ones,  and  with  many  pathogenic 
bacteria  the  lethal  dose  of  a  culture  bears  some  relation  to  the  size 
of  the  animal. 

(b)  The  point  of  inoculation.     Injections  into  the   circulation 
are  generally  more   promptly  fatal  and  require  a  smaller  dose  than 
those  into  a  serous  cavity  or  into  the  connective  tissue.     Pathogenic 
bacteria  introduced  into  the  abdominal  cavity  reach  the  circulation 
more   promptly  than   those  injected   subcutaneously.     But   certain 
microorganisms  owe  their  pathogenic  power  to  the  local  effect  about 
the  point  of  inoculation  and  the  absorption  of  toxic  products  formed 
in  the  limited  area  invaded,  and  do  not  enter  the  general  circulation, 
or  at  least  do  not  multiply  in  the   circulating  fluid,  and  quickly  dis- 
appear from  it. 

(b)  The  age  of  the  culture  injected.  Old  cultures  sometimes 
have  greater  and  sometimes  less  pathogenic  potency  than  recent  cul- 
tures. Some  kinds  of  virus  become  ' i  attenuated  "  when  kept.  But 
when  the  pathogenic  power  depends  chiefly  upon  toxic  products 
formed  during  the  growth  of  the  bacteria,  old  cultures  are,  as  a  rule, 
more  potent  than  those  recently  made. 

(d)  The  medium    in   which   the  pathogenic  bacteria  are  sus- 
pended.    Cultures  in  albuminous  media,  like  blood  serum,   are  in 
some  cases  more  potent  than  bouillon  cultures  ;  and  the  virulence  of 
several  pathogenic  bacteria  is  greatly  intensified  by  successive  cul- 
tures— by  inoculation — in  the  bodies  of  susceptible  animals.     Ogston 
found  that  pus  cocci  cultivated  in  the  interior  of  eggs  had  an  in- 
creased virulence.     According  to  Arloing,  Cornevin,  and  Thomas, 
the  activity  of  a  culture  of  the  bacillus  of  symptomatic  anthrax  is 
doubled  by  adding  one-five-hundredth  part  of  lactic  acid  to  the  cul- 
ture fluid. 

(e)  The  quantity  injected  is  evidently  an  essential  point  when 
the  result  depends  largely  upon  the  toxic  products  formed  in  the  cul- 


EXPERIMENTS   UPON   ANIMALS.  101 

tare  medium.  It  is  also  an  essential  point  when  pathogenic  bacteria 
are  injected  which  kill  susceptible  animals  in  very  minute  doses,  for 
it  has  been  shown  by  the  experiments  of  Watson  Cheyne  and  others 
that  in  the  case  of  some  of  these,  at  least,  there  is  a  limit  below 
which  infection  does  not  occur. 

Inoculated  animals  should  be  carefully  observed,  and  a  note 
made  of  every  symptom  indicating  a  departure  from  the  usual  con- 
dition of  health,  such  as  fever,  less  of  activity,  loss  of  appetite, 
weakness,  emaciation,  diarrhoea,  convulsions,  dilated  pupils,  the  for- 
mation of  an  abscess  or  a  diffuse  cellulitis  extending  from  the  point 
of  inoculation,  etc.  The  temperature  is  usually  taken  in  the  rectum. 
The  temperature  of  small  animals,  like  rabbits  and  guinea-pigs,  va- 
ries considerably  as  a  result  of  external  conditions.  In  the  rabbit 
the  normal  temperature  may  be  given  as  about  102°  to  103°  F.  ;  in 
the  guinea-pig  it  is  a  little  lower. 

In  making  a  post-mortem  examination  of  an  inoculated  animal  it 
is  best  to  stretch  it  out  on  a  board,  belly  up,  by  tying  its  legs  to  nails 
or  screws  fastened  in  the  margin  of  the  board.  When  the  abdomen 
is  dirty,  as  is  usually  the  case,  it  should  be  carefully  washed  with  a 
disinfecting  solution.  An  incision  through  the  skin  is  then  made  in 
the  median  line  the  full  length  of  the  body,  and  the  skin  is  dis- 
sected back  so  as  to  expose  the  anterior  walls  of  the  abdomen  and 
thorax.  These  cavities  are  then  carefully  opened  with  a  sterilized 
knife  or  scissors,  and  the  various  organs  and  viscera  examined.  At- 
tention should  also  be  given  to  the  appearances  at  the  point  of  in- 
oculation. To  ascertain  whether  the  microorganism  injected  has 
invaded  the  blood,  smear  preparations  should  be  made  with  blood 
obtained  from  a  vein  or  from  one  of  the  cavities  of  the  heart.  It 
will  be  well  also  to  make  a  smear  preparation  from  a  cut  surface  of 
the  liver  and  spleen.  In  the  various  forms  of  acute  septica3inia  the 
spleen  is  usually  found  to  be  enlarged.  If  but  few  microorganisms 
are  present  in  the  blood  and  tissues  they  may  escape  observation  in 
^tained  smear  preparations,  and  it  will  be  necessary  to  make  cultures 
to  demonstrate  their  presence.  A  little  blood  from  a  vein  or  from 
one  of  the  cavities  of  the  heart  is  transferred,  by  means  of  a  plati- 
num loop  (ose)  or  a  sterilized  collecting  tube  (see  page  38),  to  a 
test  tube  containing  liquefied  nutrient  gelatin  or  agar-agar,  and  an 
Esmarch  roll  tube  is  made.  This  is  put  aside  for  the  development  of 
colonies  from  any  scattered  bacteria  which  •  may  be  present.  As  a 
rule,  it  will  be  best  to  make  agar  cultures,  as  these  can  be  placed  in 
the  incubating  oven  at  35°  to  38°  C.  Stab  cultures  may  also  be 
made  and  will  serve  to  show  the  presence  of  microorganisms,  but 
will  not  give  information  as  to  how  numerous  they  may  be.  The 
roll  tube  also  has  the  advantage  of  showing  whether  there  is  a 


102  EXPERIMENTS    UPON   ANIMALS. 

mixed  infection  or  whether  a  pure  culture  of  a  single  microorganism 
is  obtained  from  the  blood.  In  the  same  way  cultures  may  be  made 
from  material  obtained  from  the  liver  or  spleen,  and  it  may  happen 
that  one  or  both  of  these  organs  contain  bacteria  when  none  are 
found  in  the  blood.  Before  passing  the  platinum  needle  or  collect- 
ing tube  into  the  organ,  the  surface,  which  has  been  more  or  less  ex- 
posed to  contamination,  should  be  sterilized  by  applying  to  it  a  hot 
spatula  ;  then  at  the  moment  of  lifting  the  spatula  the  sterilized 
needle  is  introduced  into  the  interior  of  the  organ,  and  the  blood  and 
crushed  tissue  adhering  to  it  at  once  carried  over  to  the  culture  me- 
dium. Or  blood  obtained  with  proper  precautions  from  a  vein,  a 
cavity  of  the  heart,  or  the  interior  of  the  spleen  or  liver,  may  be 
used  to  inoculate  another  animal. 

Animals  are  also  sometimes  inoculated  by  excoriating  the  cutis 
as  in  vaccination.  They  may  also,  in  rare  cases,  be  infected  by  in- 
troducing cultures  into  the  stomach,  either  mixed  with  the  food  in- 
gested or  by  injection  through  a  tube.  Infection  by  inhalation  is 
accomplished  by  causing  the  animal  to  respire  an  atmosphere,  in  a 
properly  enclosed  space,  in  which  the  pathogenic  organism  is  sus- 
pended, by  the  use  of  a  spray  apparatus  for  liquid  cultures,  or 
some  form  of  powder  blower  for  powders  containing  the  bacteria  in 
a  desiccated  condition. 

One  method  of  obtaining  a  pure  culture  of  pathogenic  bacteria 
consists  in  the  inoculation  of  susceptible  animals  with  material  con- 
taining a  pathogenic  species  in  association  with  others  which  are  not. 
When  the  blood  is  invaded  by  the  pathogenic  species  and  the  animal 
dies  from  an  acute  septicaemia,  we  may  usually  obtain  a  pure  cul- 
ture by  inoculating  a  suitable  culture  medium  with  a  minute  drop  of 
blood  taken  from  a  vein  or  from  one  of  the  cavities  of  the  heart. 
Sometimes,  however,  a  mixed  infection  occurs  and  some  other  mi- 
croorganism is  associated  in  the  blood  with  that  one  which  was  the 
immediate  cause  of  the  death  of  the  animal. 


XII. 
PHOTOGRAPHING  BACTERIA. 

WELL-MADE  photomicrographs  are  unquestionably  superior  to 
drawings  made  by  hand  as  a  permanent  record  of  morphological 
characters.  This  being  the  case,  bacteriologists  would  no  doubt  re- 
sort to  this  method  more  generally  but  for  the  technical  difficulties 
and  the  time  and  patience  required  in  overcoming  these.  Koch,  in 
his  earlier  studies,  gave  much  time  to  photographing  bacteria,  and 
with  very  remarkable  success.  In  his  work  on  "Traumatic  Infec- 
tive Diseases  "  (1878)  he  says  : 

"With  respect  to  the  illustrations  accompanying  this  work,  I 
must  here  make  a  remark.  In  a  former  paper l  on  the  examination 
and  photographing  of  bacteria  I  expressed  the  wish  that  observers 
would  photograph  pathogenic  bacteria  in  order  that  their  representa- 
tions of  them  might  be  as  true  to  nature  as  possible.  I  thus  felt 
bound  to  photograph  the  bacteria  discovered  in  the  animal  tissues  in 
traumatic  infective  diseases,  and  I  have  not  spared  trouble  in  the 
attempt.  The  smallest,  and  in  fact  the  most  interesting  bacteria, 
however,  can  only  be  made  visible  in  animal  tissues  by  staining 
them  and  by  thus  gaining  the  advantage  of  color.  But  in  this  case 
the  photographer  has  to  deal  with  the  same  difficulties  as  are  expe- 
rienced in  photographing  colored  objects — e.g.,  colored  tapestry. 
These  have,  as  is  well  known,  been  overcome  by  the  use  of  colored 
collodion.  This  led  me  to  use  the  same  method  for  photographing 
stained  bacteria,  and  I  have,  in  fact,  succeeded,  by  the  use  of  eosin- 
collodion,  and  by  shutting  off  portions  of  the  spectrum  by  colored 
glasses,  in  obtaining  photographs  of  bacteria  which  had  been  stained 
with  blue  and  red  aniline  dyes.  Nevertheless,  from  the  long  ex- 
posure required  and  the  unavoidable  vibrations  of  the  apparatus,  the 
picture  does  not  have  sharpness  of  outline  sufficient  to  enable  it  to  be 
of  use  as  a  substitute  for  a  drawing,  or,  indeed,  even  as  evidence  of 
what  one  sees.  For  the  present,  therefore,  I  must  abstain  from  pub- 
lishing photographic  representations ;  but  I  hope,  at  a  subsequent 
period  when  improved  methods  allow  a  shorter  exposure,  to  be  able 
to  remedy  this  defect/' 

1  The  paper  referred  to  is  published  in  Cohn's  "Beitrage  zur  Biologic  d.  Pflanzen." 


104  PHOTOGRAPHING   BACTERIA. 

Since  the  above  was  written  considerable  progress  has  been  made 
in  removing  the  technical  difficulties,  and  many  bacteriologists  have 
succeeded  in  making  very  satisfactory  photomicrographs.  As  speci- 
mens of  what  may  be  done  with  the  best  apparatus  and  the  highest 
degree  of  skill,  we  may  call  attention  to  the  photomicrographs  in 
the  Atlas  der  Bakterienkunde  of  Frankel  and  Pfeiffer,  and  those 
of  Roux  in  the  Annales  of  the  Pasteur  Institute.  The  writer,  also, 
has  devoted  much  time  to  making  photomicrographs  which  have 
served  as  illustrations  for  several  of  his  published  works. 

Those  who  have  had  no  practical  experience  in  making  photo- 
micrographs are  apt  to  expect  too  much  and  to  underestimate  the 
technical  difficulties.  Objects  which  under  the  microscope  give  a 
beautiful  picture,  which  we  desire  to  reproduce  by  photography,  may 
be  entirely  unsuited  for  the  purpose.  .  In  photographing  with  high 
powers  it  is  necessary  that  the  objects  to  be  photographed  be  in  a 
single  plane  and  not  crowded  together  or  overlying  each  other. 
For  this  reason  photographing  bacteria  in  sections  presents  special 
difficulties,  and  satisfactory  results  can  only  be  obtained  when  the 
sections  are  extremely  thin  and  the  bacteria  well  stained.  Even 
with  the  best  preparations  of  this  kind  much  care  must  be  taken  in 
selecting  a  field  for  photography.  It  must  be  remembered  that  the 
expert  microscopist,  in  examining  a  section  with  high  powers,  has 
his  finger  on  the  fine  adjustment  screw  and  focuses  up  and  down  to 
bring  different  planes  into  view.  He  is  in  the  habit  of  fixing  his  at- 
tention on  that  part  of  the  field  which  is  in  the  focus  and  disregard- 
ing the  rest.  But  in  a  photograph  the  part  of  the  field  not  in  focus 
appears  in  a  prominent  way  which  mars  the  beauty  of  the  picture. 
In  a  cover-glass  preparation  made  from  a  pure  culture,  when  the 
bacteria  are  well  distributed,  this  difficulty  does  not  present  itself,  as 
the  bacteria  are  all  lying  in  a  single  plane;  but  the  portion  of  the  field 
which  can  be  shown  at  one  time  is  limited  by  the  spherical  aberra- 
tion of  the  objective,  which  the  makers  do  not  seem  able  to  overcome 
in  high-power  lenses  of  wide  angle,  at  least  not  without  loss  of  de- 
fining power. 

Usually  preparations  of  bacteria  are  stained  for  photography, 
but  with  some  of  the  larger  forms,  such  as  the  anthrax  bacillus, 
very  satisfactory  photomicrographs  may  be  made  from  unstained 
preparations.  In  this  case  a  small  quantity  of  a  recent  culture  is 
put  upon  a  slide,  covered  with  a  thin  cover  glass,  and  placed  at  once 
upon  the  stage  of  the  microscope.  The  main  difficulty  to  be  encoun- 
tered results  from  the  change  of  location  of  the  suspended  bacteria 
resulting  from  the  pressure  of  the  objective  in  focussing.  Motile 
bacteria,  of  course,  cannot  be  photographed  in  this  way  without  first 
arresting  their  movements  by  means  of  some  germicidal  agent ; 


PHOTOGRAPHING    BACTERIA.  105 

and  in  general  it  will  be  found  more  satisfactory  to  fix  the  micro- 
organisms to  be  photographed  to  a  slide  or  cover  glass  by  desiccation 
and  heat,  and  to  stain  them  with  one  of  the  aniline  colors. 

Objects  which  are  opaque  cannot  be  photographed  by  transmitted 
light,  and  objects  which  have  a  deep  orange  or  red  color  are  practi- 
cally opaque  for  the  actinic  rays  which  are  at  the  violet  end  of  the 
spectrum.  Such  objects  simply  intercept  the  light,  but  this  gives 
the  outlines,  and,  where  there  are  no  details  of  structure,  is  all  that 
is  required  to  illustrate  the  form  and  mode  of  grouping.  Softer  and 
more  satisfactory  photomicrographs  of  bacteria  are  made  when  the 
staining  is  not  such  as  to  entirely  arrest  the  actinic  rays.  Among 
the  aniline  colors  Bismarck  brown  and  vesuvin  are  the  most  suitable, 
care  being  taken,  with  the  larger  bacteria  especially,  not  to  make 
the  staining  too  intense.  Objects  which  are  transparent  for  the  ac- 
tinic rays,  or  nearly  so,  give  a  very  feeble  photographic  image,  or 
none  at  all,  on  account  of  the  want  of  contrast  in  the  impression 
made  upon  the  sensitive  plate.  This  is  the  case  when  we  attempt  to 
photograph,  by  ordinary  white  light,  objects  which  are  stained  violet 
or  blue.  But  this  want  of  contrast  in  the  negative  can  be  overcome 
by  the  use  of  specially  prepared  plates  and  colored  screens  of  glass 
interposed  between  the  object  and  the  source  of  light.  The  so-called 
orthochromatic  plates  are  more  sensitive  to  the  rays  toward  the  red 
end  of  the  spectrum  than  ordinary  plates.  They  are  prepared  by 
treating  the  plates  with  a  solution  of  eosin,  of  erythrosin,  or  of  rose 
bengal  (Vogel),  and  may  now  be  purchased  in  this  country  from 
dealers  in  dry  plates.  If  we  shut  off  the  violet  rays  by  the  use  of  a 
yellow  screen,  objects  having  a  yellow  or  orange  color  may  be  pho- 
tographed upon  orthochromatic  plates,  although  the  time  of  exposure 
will  be  quite  long  owing  to  the  comparatively  feeble  actinic  power 
of  the  yellow  rays. 

We  may  also  make  photomicrographs  of  objects  stained  with 
methylene  blue  or  with  fuchsin,  because  objects  stained  with  these 
colors  are  opaque  for  the  rays  from  the  red  end  of  the  spectrum,  and 
sufficiently  so  with  yellow  light  to  give  a  good  photographic  con- 
trast. Frankel  and  Pfeiffer  recommend  the  use  of  a  green  light-fil- 
ter (green  glass  screen)  for  all  preparations  stained  with  methyl  vio- 
let, fuchsin,  or  methylene  blue;  and  for  brown-stained  preparations  a 
pure  blue  light.  The  writer  has  been  in  the  habit  of  using  a  yellow 
glass  screen  for  fuchsin-stained  preparations,  and  has  had  excellent 
results,  but  the  time  of  exposure  is  necessarily  long.  A  yellow  glass 
screen  may  be  prepared  by  dissolving  tropa3olin  in  negative  varnish, 
and  pouring  this  upon  a  clean  glass  slide,  where  it  is  permitted  to 
dry. 

To  show  bacteria  in  photographs  in  a  satisfactory  manner  we 


106  PHOTOGRAPHING   BACTERIA. 

require  an  amplification  of  five  hundred  to  one  thousand  diameters  ; 
and  as  it  is  often  desirable  to  make  comparisons  as  to  the  dimen- 
sions of  microorganisms  which  resemble  each  other  in  form,  it  is 
best  to  adopt  a  standard  amplification.  The  writer  has  himself 
adopted,  and  would  recommend  to  others,  a  standard  amplification 
of  one  thousand  diameters.  This  is  about  as  high  a  magnifying 
power  as  we  can  get  with  satisfactory  definition,  or  as  we  require, 
and  it  is  a  convenient  number  when  measurements  are  made  from 
the  photograph.  The  beginner,  after  having  put  his  apparatus  in 
position,  should  focus  the  lines  of  a  stage  micrometer  upon  the 
screen  with  the  optical  apparatus  which  he  proposes  to  use  ;  then  by 
moving  the  screen  forward  or  back  as  required,  and  carefully  focus- 
sing the  lines,  he  will  ascertain  what  is  the  position  of  the  screen  for 
exactly  one  thousand  diameters.  If  the  stage  micrometer  is  ruled 
with  lines  which  are  one  one-thousandth  of  an  inch  apart,  it  is  evi- 
dent that  when  projected  upon  the  screen  they  should  be  one  inch 
apart  to  make  the  amplification  one  thousand  diameters.  But  it 
must  be  remembered  that  any  change  in  the  position  of  the  optical 
combination  will  change  the  amplification.  If,  therefore,  the  cover 
correction  of  the  objective  is  changed,  or  the  position  of  the  eyepiace 
— if  one  is  used — it  will  be  necessary  to  again  adjust  the  distance  of 
the  screen. 

Apparatus  required. — A  first-class  immersion  objective  of  one- 
twelfth  of  an  inch  or  higher  power,  a  substantial  stand  which  can  be 
placed  in  a  horizontal  position,  and  a  camera  which  can  be  coupled 
with  the  microscope  tube,  are  the  essential  pieces  of  apparatus.  If 
sunlight  is  to  be  used  a  heliostat  will  also  be  required. 

The  oil-immersion  objectives  of  any  good  maker  may  be  used, 
but  the  apochromatic  objectives  and  projection  eyepieces  of  Carl 
Zeiss,  of  Jena,  are  especially  to  be  recommended.  Indeed,  those  who 
can  afford  it  will  do  well  to  get  Zeiss'  complete  apparatus,  which 
includes  a  stand  having  a  mechanical  stage  and  a  camera  mounted 
upon  a  metal  frame  conveniently  provided  with  focussing  appliances, 
etc.  However,  good  work  may  be  done  with  less  expensive  appa- 
ratus. 

The  stand  should  be  substantial  and  well  made,  with  a  delicate, 
fine  adjustment.  A  mechanical  stage  is  not  essential,  but  is  a  great 
convenience  in  finding  and  adjusting  to  the  centre  of  the  screen  a 
satisfactory  field  to  photograph.  The  substage  should  be  provided 
with  a  good  apochromatic  condenser,  and  with  appliances  for  moving 
the  condensing  lens  forward  and  back  and  for  centring  it,  with  dia- 
phragms, etc. 

By  the  use  of  a  high-power  objective,  like  the  one-eighteenth-inch 
oil-immersion  of  Zeiss,  the  desired  amplification  may  be  obtained 


PHOTOGRAPHING    BACTERIA.  107 

without  the  use  of  an  eyepiece  ;  and,  as  a  rule,  it  is  best  not  to  use 
an  ordinary  eyepiece  to  secure  increased  amplification,  as  this  is  ob- 
tained at  the  expense  of  definition.  But  an  amplifier  may  be  used  in 
the  tube  of  the  microscope,  as  first  recommended  by  Woodward.  In 
this  case  the  amplifier  must  be  carefully  adjusted  with  reference  to 
the  distance  of  the  screen,  to  secure  the  best  possible  definition. 

The  projection  eyepieces  of  Zeiss  are  constructed  especially  for 
photography  and  possess  a  decided  advantage.  By  the  use  of  his 
three-millimetre  apochromatic  oil-immersion  objective  and  projec- 
tion eyepiece  No.  3  we  may  obtain  an  amplification  of  one  thousand 
diameters  with  excellent  definition. 

Light. — Sunlight  is  in  many  respects  the  most  satisfactory  for 
photography,  but  has  the  disadvantage  that  it  is  not  always  available. 
In  some  sections  of  the  country  weeks  may  pass  without  a  single 
clear  day  suitable  for  making  photomicrographs.  In  addition  to  the 
uncertainty  arising  from  cloudy  weather,  we  have  to  contend  with 
the  fact  that  the  sun  is  only  available  for  use  with  a  heliostat  for  a 
limited  time  during  each  day,  and  that  this  time  is  greatly  restricted 
in  Northern  latitudes  during  the  winter  months.  When  sunlight  is 
to  be  employed  the  microscope  and  camera  must  be  set  up  in  a  room 
having  a  southern  exposure  on  a  line  corresponding  with  the  true 
meridian  of  the  place.  The  heliostat  is  placed  outside  the  window  in 
such  a  position  that  when  properly  adjusted  the  light  of  the  sun  will 
fall  upon  the  condenser  attached  to  the  substage  of  the  microscope. 
The  condenser  must  be  carefully  centred,  so  that  the  circle  of  light 
falling  upon  the  screen  shall  be  uniform  in  intensity  and  outline.  . 

The  calcium,  magnesium,  or  electric  light  may  be  used  as  a  sub- 
stitute for  sunlight,  but  they  are  all  rather  expensive,  unless,  in  the 
case  of  the  electric  light,  a  suitable  current  is  available  without  the 
expense  of  generating  it  for  the  special  purpose  in  view.  The  writer 
has  obtained  very  good  results  with  the  calcium  light,  but  has  no  ex- 
perience in  the  use  of  the  electric  light.  Woodward,  as  a  result  of 
extended  experiments,  arrived  at  the  conclusion  that  "the  electric 
light  is  by  far  the  best  of  all  artificial  lights  for  the  production  of 
photomicrographs. "  He  used  a  Grove  battery  of  fifty  elements  to 
generate  the  current,  and  a  Duboscq  lamp.  The  current  from  a 
dynamo  would  no  doubt  be  much  cheaper  and  more  conveniently 
used,  if  an  electric-lighting  plant  was  in  the  vicinity. 

The  apparatus  shown  in  Fig.  73  was  designed  by  Mr.  Pringle  for 
the  use  of  the  calcium  light.  It  will  serve  to  illustrate  the  arrange- 
ment of  the  microscope  and  camera  in  connection  with  any  other 
light  as  well.  An  oil  lamp  may  be  placed  in  the  position  of  the  oxy- 
hydrogen  burner  ;  or,  if  sunlight  is  to  be  employed,  a  heliostat  will 
be  placed  in  the  same  position. 


108 


PHOTOGRAPHING    BACTERIA. 


When  a  colored  screen  is  used  this  may  be  placed  either  before 
or  behind  the  condensing  lens — we  prefer  to  place  it  behind,  although 


i 


Neuhauss  has  shown  that  it  makes  no  difference  in  the  length  of  the 
exposure. 

We  cannot  in  the  present  volume  give  full  details  with  reference 


PHOTOGRAPHING    BACTERIA.  109 

to  the  technique  of  making  photomicrographs,  but  append  an  account 
of  a  form  of  apparatus  which  we  have  used  with  great  satisfaction  : 

"Photomicrography  by  Gaslight. — Those  who  have  had  much  experience 
in  making  photomicrographs  will  agree  with  me  that  one  of  the  most  essen- 
tial elements  of  success  is  the  use  of  a  suitable  source  of  illumination. 

"  Without  question  the  direct  light  of  the  sun,  reflected  in  a  right  line  by 
the  mirror  of  a  heliostat,  is  the  most  economical  and,  in  some  respects,  the 
most  satisfactory  light  that  can  be  used.  But  we  cannot  command  this  light 
at  all  times  and  places,  and  it  often  happens  that,  when  we  are  ready  to  de- 
vote a  day  to  making  photomicrographs,  the  sun  is  obscured  by  clouds  or 
the  atmosphere  is  hazy.  Indeed,  in.  some  latitudes  and  at  certain  seasons  of 
the  year  a  suitable  day  for  the  purpose  is  extremely  rare.  The  use  of  sun- 
light also  requires  a  room  having  a  southern  exposure  and  elevated  above  all 
surrounding  buildings  or  other  objects  by  which  the  direct  rays  of  the  sun 
would  be  intercepted.  For  these  reasons  a  satisfactory  artificial  light  is  ex- 
tremely desirable. 

"  The  oxyhydrogeii  lime  light,  the  magnesium  light,  and  the  electric  arc 
light  have  all  been  employed  as  a  substitute  for  the  light  of  the  sun,  and  all 
give  satisfactory  results.  I  have  myself  made  rather  extensive  use  of  the 
'limelight,'  and  think  it  the  best  substitute  for  solar  light  with  which  I 
am  familiar.  But  to  use  it  continuously,  day  after  day,  is  attended  with 
considerable  expense,  and  the  frequent  renewal  of  the  -supply  of  gas  which 
it  calls  for  is  an  inconvenience  which  one  would  gladly  dispense  with. 

"  These  considerations  have  led  some  microscopists  to  use  an  oil  lamp  as 
the  source  of  illumination,  and  very  satisfactory  photomicrographs  with 
comparatively  high  power  have  been  made  with  this  cheap  and  convenient 
light.  But  in  my  experience  the  best  illumination  which  I  have  been  able 
to  secure  with  an  oil  lamp  has  called  for  very  long  exposures  when  working 
with  high  powers,  and,  as  most  of  my  photomicrographs  of  bacteria  are 
made  with  an  amplification  of  one  thousand  diameters,  I  require  a  more 
powerful  illumination  than  I  have  been  able  to  secure  in  this  way.  And 
especially  so  because  of  the  fact  that  a  colored  screen  must  be  interposed, 
which  shuts  off  a  large  portion  of  the  actinic  rays,  on  account  of  the  staining 
agent  usually  employed  in  making  my  mounts.  The  most  satisfactory 
staining  agents  for  the  bacteria  are  an  aqueous  solution  of  fuchsin,  or  of 
methylene  blue,  or  of  gentian  violet;  and  all  of  these  colors  are  so  nearly 
transparent  for  the  actinic  rays  at  the  violet  end  of  the  spectrum  tbat  a 
satisfactory  photographic  contrast  cannot  be  obtained  unless  we  shut  off 
these  rays  by  a  colored  screen. 

"  I  am  in  the  habit  of  using  a  yellow  screen  for  rny  preparations  stained 
with  fuchsin  or  methylene  blue,  and  have  obtained  very  satisfactory  results 
with  the  orthochromatic  plates  manufactured  by  Carbutt,  of  Philadelphia, 
and  a  glass  screen  coated  with  a  solution  of  tropaeolin  dissolved  in  gelatin. 

"  But  with  such  a  screen,  which  shuts  off  a  large  portion  of  the  actinic 
light  and  increases  the  time  of  exposure  three-  or  fourfold,  the  use  of  an 
oil  lamp  becomes  impracticable  with  high  powers,  on  account  of  the  feeble^ 
ness  of  the  illumination. 

"These  considerations  have  led  me  to  experiment  with  gaslight,  and  the 
simple  form  of  apparatus  which  I  am  about  to  describe  is  the  result  of  these 
experiments.  I  have  now  had  the  apparatus  in  use  for  several  months, 
during  which  time  I  have  made  a  large  number  of  very  satisfactory  photo- 
micrographs of  bacteria  from  fuchsin-stained  preparations  with  an  amplifica- 
tion of  one  thousand  diameters.  My  photographs  have  been  made  with  the 
three-millimetre  oil-immersion  apochromatic  objective  of  Zeiss  and  his  pro- 
jection eyepiece  No.  3.  I  use  a  large  Powell  and  Lealand  stand,  upon  the 
substage  of  which  I  have  fitted  an  Abbe  condenser.  The  arrangement  of 
the  apparatus  will  be  readily  understood  by  reference  to  the  accompanying 
figure. 

"A  is  the  camera,  which  has  a  pyramidal  bellows  front  supported  by  the 


110 


PHOTOGRAPHING   BACTERIA. 


heavy  block  of  wood  B ;  this  can  be  pushed  back  upon  the  baseboard  which 
supports  it,  so  as  to  allow  the  operator  to  place  his  eye  at  the  eyepiece  of  the 
microscope.  When  it  is  brought  forward  an  aperture  of  the  proper  size  ad- 
mits the  outer  extremity  of  the  eyepiece  and  shuts  off  all  light  except  that 
coming  through  the  objective.  C  is  the  microscope,  and  D  the  Abbe  con- 
denser, supported  upon  the  substage.  E  is  a  thick  asbestos  screen  for  pro- 
tecting the  microscope  from  the  heat  given  off  by  the  battery  of  gas  burners 
F.  This  asbestos  screen  has  an  aperture  of  proper  dimensions  to  admit  the 
light  to  the  condensjr  D.  The  gas  burners  are  arranged  in  a  series,  with 
the  flat  portion  of  the  flame  facing  the  aperture  in  the  asbestos  screen  E. 
The  concave  metallic  mirror  G-  is  properly  placed  to  reflect  the  light  in  the 
desired  direction.  I  have  not  found  any  advantage  in  the  use  of  a  condens- 
ing lens  other  than  the  Abbe  condenser  upon  the  substage  of  the  microscope. 
The  focussing  is  accomplished  by  means  of  the  rod  I,  which  carries  at  one 
extremity  a  grooved  wheel,  H,  which  is  connected  with  the  fine  adjustment 
screw  of  the  microscope  by  means  of  a  cord. 

I'  The  focussing  wheel  J  may  be  slipped  along  the  rod  I  to  any  desired 
position,  and  is  retained  in  place   by  a  set  screw.     The   rod  I  is  supported 


FIG.  74. 

above  the  camera  by  arms  depending  from  the  ceiling,  or  by  upright  arms 
attached  to  the  baseboard. 

"  I  have  lost  many  plates  from  a  derangement  of  the  focal  adjustment 
resulting  from  vibrations  caused  by  the  passing  of  loaded  wagons  in  the 
street  adjoining  the  laboratory  in  which  I  work.  This  has  been  overcome 
to  a  great  degree  by  placing  soft  rubber  cushions  under  the  whole  appa- 
ratus. " l 

I  have  recently  (1895)  seen  a  gaslight  which  I  believe  would  prove 
to  be  a  valuable  substitute  for  ordinary  street  gas,  and  I  judge  that, 
owing  to  its  superior  brilliancy,  a  single  jet  would  suffice  to  replace  the 
five  burners  in  a  linear  series  which  are  shown  in  the  above  figure. 
The  gas  referred  to  is  acetylene,  which  may  now  be  obtained  in  a 
liquid  form  in  strong  metal  cylinders.  Reference  has  already  been 
made  to  the  use  of  an  oil  light,  and  for  low  powers  an  ordinary  lamp 
with  a  flat  wick  may  be  used.  That  bacteria  may  be  successfully 
photographed,  with  an  amplification  of  one  thousand  diameters,  by 
means  of  an  oil  lamp  is  shown  by  the  beautiful  photomicrographs 
made  by  Capt.  VV.  C.  Borden,  Assistant  Surgeon  U.  S.  Army. 
At  my  request  Dr.  Borden  has  prepared  the  following  detailed 
account  of  his  method  : 

JFrom  Johns  Hopkins  University  Circulars,  vol.  ix.,  No.  81,  p.  72. 


PHOTOGRAPHING   BACTERIA.  Ill 


DESCRIPTION     OF     APPARATUS     FOR     PHOTOMICROGRAPHY     BY     OIL 

LIGHT. 

The  apparatus  consists  of  a  camera,  hung-  in  a  vertical  position,  of  a 
microscope  with  substage  condensers,  suitable  objectives  and  projection 
oculars,  and  a  Laverne  tri-wick,  oil  stereopticon  with  the  projection  objec- 
tive removed. 

The  Light. — After  trying  all  kinds  of  lamps,  I  found  that  the  best  illu- 
mination could  be  obtained  by  using  a  tri-wick  stereopticon  with  the  pro- 
jection objective  removed,  the  middle  wick  only  being  lighted.  The  large 
four-inch  condensers  serve  to  concentrate  the  light,  while  the  double  lantern 
body  prevents  the  radiation  of  heat  to  the  microscope  and  shuts  off  all  radiat- 
ing-light.  Consequently  the  microscope  does  not  become  heated,  and  if  the 
room  is  darkened  the  absence  of  extraneous  light  greatly  aids  in  focussing  on 
the  camera  screen.  The  oil  light  itself  is  quite  yellow  and  so  nearly  mono- 
chromatic that  with  orthochromatic  plates  a  color  screen  is  seldom,  if  ever, 
required.  After  experimenting  by  taking  photographs  with  and  without  a 
screen,  I  have  found  no  particular  difference  in  result  even  when  photo- 
graphing difficult  bacteria,  and  now  seldom  use  one.  If  a  screen  is  used  a 
solution  of  bichromate  of  potash  and  sulphate  of  copper  in  dilute  ammonia 
water  placed  in  a  trough  between  the  lantern  and  microscope  gives  excellent 
results  and  does  not  materially  lengthen  the  time  of  exposure.  The  lantern 
is  placed  about  twelve  inches  in  front  of  the  microscope  and  with  its  central 
long  axis  in  a  plane  which  extends  through  the  centre  of  the  microscope 
mirror,  the  substage  condenser,  the  objective,  ocular  and  centre  of  camera. 

Microscope. — The  microscope  is  used  in  the  upright  position.  I  have 
used  this  position  rather  than  the  horizontal  for  several  reasons.  The 
microscope  is  used  on  the  work-table  in  an  upright  position,  and  in  working 
when  an  object  is  found  which  it  is  desired  to  photograph,  the  microscope 
without  changing  adjustments  has  only  to  be  carried  to  the  photomicro- 
graphic  apparatus,  placed  in  position,  correct  adjustments  of  light  made,  the 
camera  racked  into  contact  and  the  exposure  made.  With  a  conveniently 
placed  dark  room  the  whole  operation  will  occupy  but  a  few  minutes.  The 
upright  position  is  necessitated  when  liquid  preparations,  as  colonies  of 
bacteria  floating  on  liquefied  gelatin,  are  to  be  photographed,  or  when  the 
microscope  is  used  for  clinical  photomicrography,  as  in  photographing  uri- 
nary deposits  in  urine,  blood  corpuscles  in  Thoma  blood  counter,  etc.  In 
bacteriological  work  where  the  bacteria  are  stained  on  the  cover  and  after 
mounting  the  balsam  is  not  quite  dry,  the  cover  is  apt  to  slip  if  the  micro- 
scope is  used  horizontally,  but  this  does  not  occur  with  the  microscope  placed 
vertically.  The  horizontal  position  and  long  extension  of  camera  is  neces- 
sary for  certain  work,  particularly  where  large  pictures  (i.e.,  over  four 
inches  in  diameter)  have  to  be  taken,  or  where  it  is  desired  to  obtain  high  am- 
plification by  extension  of  camera  rather  than  by  high  eyepiecing,  or  in 
photographing  test  diatoms  with  very  high  amplifications.  For  practical 
work,  however,  up  to  amplifications  of  one  thousand  diameters,  and  for 
photographs  for  illustration  or  reproduction,  which  are  seldom  required  of 
over  three  and  one-half  or  four  inches  in  diameter,  the  upright  position  is 
much  to  be  preferred  on  account  of  its  ease  of  application  and  its  practical 
advantages. 

Camera. — The  upright  position  of  the  microscope  necessitates  a  similar 
position  for  the  camera.  To  allow  easy  working,  the  camera  is  hung  on  a 
rack- work  attached  to  a  rigid  upright.  "  The  upright  is  placed  to  the  right  of 
the  microscope  so  that  it  will  be  out  of  the  way  while  working. 

Both  the  upper  and  the  lower  ends  of  the  camera  are  movable  on  the 
rack-work.  The  upper  end,  which  carries  the  screen  and  plate-holder,  is 
movable,  in  order  that  different  amplifications  within  limits  can  be  gotten 
with  the  same  objective.  The  lower  end  is  movable  that  it  may  be  racked 


112  PHOTOGRAPHING   BACTERIA. 

up  and  out  of  the  \vay  and  allow  the  operator  to  manipulate  the  microscope 
before  attaching1  the  camera.  The  bellows  has  an  extension  of  two  feet, 
measured  from  the  eyepiece  of  the  microscope  to  the  focussing  screen.  This, 
with  a  two-millimeter  objective  and  projection  ocular  4,  gives  an  amplifica- 
tion of  one  thousand  diameters.  With  less  extension  of  bellows  and  lower 
objectives  amplifications  ranging  down  to  ten  diameters  may  be  obtained. 
In  focussing,  the  operator  can,  by  standing1,  observe  the  image  on  the  screen 
with  a  focussing1  glass  and  manipulate  the  fine  adjustment  of  the  microscope 
with  his  hand  without  using-  a  focussing'  rod,  though  a  suitable  focussing  rod 
can  be  easily  fastened  to  the  camera  upright  if  desired. 

Setting  Up  the  Apparatus. — The  camera  being  hung  011  the  rack-work 
the  microscope  is  placed  beneath  it,  a  stage  micrometer  is  placed  on  the  stage 
and  a  medium-power  objective  and  eyepiece  attached  to  the  microscope. 
Light  is  reflected  from  the  lantern  upon  the  object  by  the  mirror  of  the 
microscope,  the  observer  accurately  centres  the  micrometer,  then  removing 
the  working1  eyepiece  a  projection  ocular  is  inserted,  the  camera  racked 
down,  and  with  the  image  of  the  micrometer  projected  on  the  camera  screen 
the  microscope  is  moved  in  such  position  that  the  centre  of  the  micrometer 
image  is  exactly  in  the  centre  of  the  screen.  This  position  of  the  microscope 
is  marked  once  for  all,  and  whenever  afterward  the  microscope  is  placed  in 
the  same  place  the  centre  of  the  object  will  be  projected  on  the  centre  of  the 
screen.  To  correctly  place  the  lantern,  a  lower-power  objective  is  used,  to- 
gether with  a  high-power  (Abbe)  condenser.  The  objective  is  accurately 
focussed  on  the  lines  of  the  stage  micrometer;  by  adjusting  the  substage  con- 
denser a  clear  image  of  the  lamp  flame  is  projected  on  the  plane  of  the  ob- 
ject (micrometer)  and  the  lantern  is  moved  to  such  position  that  the  image 
will  be  central.  If  the  camera  is  attached,  the  image  will  appear  central 
on  the  focussing  screen. 

This  position  of  the  lantern,  like  that  of  the  microscope,  should  be  fixed. 

To  Photograph. — In  photographing-  by  oil  light  with  all  but  the  lowest 
powers  some  form  of  substage  condenser  is  necessary.  This  is  due  to  the  fact 
that  the  source  of  light  must  always  be  focussed  on  the  object  in  order  to  give 
proper  definition.  In  working  with  the  objectives  of  four  millimetres  or 
lower,  it  will  be  found  advantageous  to  use  objectives  of  lower  power  as 
substage  condensers,  for  it  will  be  found  that  if  placed  in  the  substage  for 
ordinary  work  they  greatly  improve  the  definition  of  objects.  In  fact  it  may 
be  laid  down  as  a  general  rule  that  whatever  with  a  given  light  gives  the 
best  definition  to  the  observer's  eye  will  give  the  sharpest  photographic  image. 
Consequently,  in  high-power  work  where  a  condenser  is  used  it  will  seldom 
be  necessary  to  change  the  microscope  attachments  when  a  photograph  has 
to  be  taken ;  for  in  bacteriological  work  the  Abbe  condenser  which  gives 
good  definition  will,  when  properly  adjusted,  give  good  photographic  defi- 
nition also,  statements  to  the  contrary  notwithstanding. 

To  photograph,  place  the  microscope  and  lantern  in  position,  light  the 
centre  wick  of  the  lamp,  place  a  ground  glass  between  the  lamp  and  camera, 
and  focus  the  objective  accurately  on  the  object.  The  ground  glass  is  used 
only  to  reduce  the  light  which  might  otherwise  injure  the  observer's  eye. 

The  ground  glass  is  then  removed,  a  fine  wire  screen  placed  close  against 
the  front  of  the  lantern  condenser,  and  by  means  of  the  substage  condenser 
an  image  of  the  screen  is  projected  accurately  on  the  object.  This  is  very 
important,  for  it  is  necessary  that  the  light  should  be  accurately  focussed  on 
the  object  in  order  to  produce  sharp  definition.  After  focussing  the  light,  the 
screen  is  removed  and  an  opal  glass  is  put  in  its  place.  On  looking  through 
the  eyepiece  a  clear  sharp  image  of  the  object  will  be  seen.  If  an  Abbe  con- 
denser is  used  the  iris  diaphragm  of  the  condenser  should  now  be  carefully 
opened  and  closed  until  such  an  aperture  is  obtained  that  to  the  observer's 
eye  the  object  appears  to  the  best  advantage.  The  opal  glass  is  now  removed, 
the  camera  attached  to  the  microscope,  and  the  projected  image  focussed  on 
the  camera  screen,  preparatory  to  exposure. 


PHOTOGRAPHING  BACTEPtlA.  113 

Plate  Used. — Orthochromatic  plates  only  should  be  used.  Of  these  I  use 
the  Cramer  rapid,  isochromatic  plate  exclusively.  With  these  when  photo- 
graphing bacteria  and  using  an  amplification  of  one  thousand  diameters  the 
exposure  will  vary  from  one  and  one-half  to  three  minutes,  two  minutes 
being  about  the  average. 

It  is  with  these  plates  that  I  have  found  a  color  screen  unnecessary,  and 
since  using  them  I  have  had  110  difficulty  in  photographing  bacteria,  for  they 
are  particularly  sensitive  to  the  yellow-colored  oil  light. 

Possibly  other  makes  of  orthochromatic  plates  might  be  found  to  work 
equally  well,  but  the  oil  light  works  so  very  well  with  the  Cramer  isochro- 
matic that  I  have  had  no  desire  to  try  others. 

Development. — For  development,  I  have  obtained  best  results  with  for- 
mulas in  which  hydrochinon  either  alone  or  with  some  other  developing 
agent  is  used.  The  following  gives  excellent  results,  and  I  prefer  it  to  other 
developers  as  it  gives  good  clear  negatives  of  sufficient  contrast  and 
gradation : 

No.  i. 

Water,       .......  10  ounces. 

Sodium  sulphite,        .  ...        1  ounce. 

Potassium  bromide,         .  .  ...  10  grains. 

Hydrochinon,  .  .  ...  30  grains. 

Metol,        ...  40  grains. 

No.  2. 

Water,  .  ......      10  ounces. 

Sodium  carbonate,  ...  .          300  grains. 

Use  equal  parts  of  No.  1  and  No.  2. 

Development  should  be  continued  until  sufficient  density  is  obtained.  In- 
tensification should  be  rarely  required,  for  with  proper  exposure  and  develop- 
ment a  good  negative  can  usually  be  obtained.  If  intensification  is  neces- 
sary, after  fixing  and  washing  the  plate,  I  prefer  to  use  a  saturated  aqueous 
solution  of  bichloride  of  mercury,  followed  by  washing,  the  application  of 
dilute  ammonia  water,  and  a  final  washing. 

Students  who  desire  to  perfect  themselves  in  the  art  of  making 
photomicrographs  are  advised  to  first  make  themselves  familiar  with 
the  technique  of  photography  with  a  landscape  or  portrait  camera, 
and  not  to  undertake  the  more  difficult  task  of  photographing  bac- 
teria until  they  know  how  to  make  a  good  negative  and  to  judge 
whether  an  exposure  has  been  too  long  or  too  short,  etc. 
8 


PLATE   I. 

PHOTOMICROGRAPHS    OF    BACTERIA    MADE    BY    GASLIGHT, 

FIG.  1. — Streptococcus  cadaveris,  from  a  culture  in  aguacoco;  stained 
with  fuchsiu.  x  1,000.  (Sternberg.) 

FIG.  2. — Streptococcus  Havaniensis.  X  1,000.  From  a  photomicro- 
graph. (Sternberg.) 

FIG.  3. — Bacillus  cuniculicida  Havaniensis,  from  peritoneal  cavity  of 
inoculated  rabbit,  showing  leucocytes  containing  bacilli  and  free  bacilli; 
stained  with  fuchsin.  X  1,000.  (Sternberg.) 

FIG.  4. — Bacillus  cadaveris,  smear  preparation  from  yellow-fever 
liver  kept  for  forty-eight  hours  in  an  antiseptic  wrapping  (Havana, 
1889);  stained  with  fuchsin.  x  1,000.  (Sternberg.) 

Note. — All  of  the  above  photomicrographs  were  made  with  the  three- 
millimetre  apochromatic  horn.  ol.  iin.  objective  and  projection  eye-piece 
of  Zeiss. 


PLATE   II. 

PHOTOGRAPHS    OF    COLONIES    (iN    ESMARCH     ROLL    TUBES)    AXD     OF     TEST- 
TUBE    CULTURES. 

FIG.  1. — Colonies  of  Bacillus  leporis  lethalis,  in  gelatin  roll  tube, 
end  of  forty-eight  hours  at  room  temperature.  X  5.  (Sternberg.) 

FIG.  2. — Colonies  of  Bacillus  coli  similis  in  gelatin  roll  tube,  end  of 
twenty-four  hours  at  22°  C.  x  10.  (Sternberg.) 

FIG.  3. — Stick  culture  of  Bacillus  coli  similis  in  nutrient  gelatin,  end 
of  seven  days  at  20°  C.  (Sternberg.) 

FIG.  4. — Stick  culture  of  Bacillus  intestinus  motilis  in  nutrient  gel- 
atin, end  of  four  days  at  22°  C.  (Sternberg.) 

FIG.  5. — Stick  culture  of  Bacillus  leporis  lethalis  in  nutrient  gelatin, 
end  of  eight  days  at  22°  C.  (Sternberg.) 

FIG.  6. — Stick  culture  of  Micrococcus  tetragenus  versatilis  in  nutrient 
gelatin,  end  of  two  weeks  at  22°  C.  (Sternberg.) 

FIG.  7. — Colonies  of  Bacillus  cuniculicida  Havaniensis  in  gelatin  roll 
tube,  end  of  forty-eight  hours  at  21°  C.  X  10.  (Sternberg.) 

FIG.  8. — Colonies  of  Bacillus  coli  communis  in  gelatin  roll  tube,  end 
of  forty-eight  hours  at  22°  C.  X  10.  (Sternberg.) 


PLATE  I. 

STERNBERG'S  BACTERIOLOGY. 


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PHOTOMICROGRAPHS  BY  GAS  LIGHT. 


PLATE  II. 

STERNBERG'S  BACTERIOLOGY. 


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Figr.  3. 


Fig;.  4. 


Figr.  2. 


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Fig;.  8. 


COLONIES  AND  TEST  TUBE  CULTURES. 


PART   SECOND. 


GENERAL  BIOLOGICAL  CHARACTERS: 

INCLUDING   AN   ACCOUNT   OF   THE   ACTION   OF   ANTISEPTICS 

AND   GERMICIDES. 

I.  STRUCTURE,   MOTIONS,  REPRODUCTION.     II.    CONDITIONS  OF  GROWTH. 

III.   MODIFICATIONS  OF  BIOLOGICAL  CHARACTERS.     IV.  PRODUCTS  OF 

VITAL  ACTIVITY.  V.  PTOMAINES  AND  TOXALBUMINS.    VI.  INFLUENCE 

OF  PHYSICAL  AGENTS.    VII.  ANTISEPTICS  AND  DISINFECTANTS 

—GENERAL  ACCOUNT  OF  THE  ACTION  OF.    VIII.  ACTION  OF 

GASES  AND  OF  THE  HALOID  ELEMENTS  UPON  BACTERIA. 

IX.  ACTION  OF  ACIDS  AND  ALKALIES.    X.  ACTION  OF 
VARIOUS  SALTS.  XI.  ACTION  OF  COAL-TAR  PRO- 
DUCTS,  ESSENTIAL  OILS,  ETC.     XII.  AC- 
TION OF  BLOOD  SERUM  AND  OTHER  OR- 
GANIC LIQUIDS.    XIII.  PRACTICAL 
DIRECTIONS  FOR  DISINFECTION. 


PART    SECOND. 


I. 
STRUCTURE,    MOTIONS,   REPRODUCTION. 

THE  bacteria  are  unicellular  vegetable  organisms,  and  consist  of 
a  cell  membrane  enclosing  transparent  and  apparently  structureless 
protoplasm.  The  very  varied  biological  characters  which  distin- 
guish different  species  make  it  evident,  however,  that  there  are  es- 
sential differences  in  the  living  cell  contents,  although  these  differ- 
ences are  not  revealed  by  our  optical  appliances.  And  among  the 
bacteria,  as  in  the  cells  of  higher  plants  and  animals,  the  peculiar 
biological  characters  of  a  species  are  transmitted  to  the  cellular  pro- 
geny of  each  individual  cell.  These  characters  are,  however,  sub- 
ject to  various  modifications  as  a  result  of  differing  conditions  of 
environment,  as  is  the  case  with  plants  and  animals  higher  in  the 
scale  of  existence,  and  in  this  way  more  or  less  permanent  varieties 
are  produced.  It  is  probable  that  among  these  lowly  plants  species 
are  evolved  more  quickly,  as  a  result  of  the  laws  of  natural  selec- 
tion, in  the  struggle  for  existence,  than  among  those  of  more  com- 
plex organization.  Still,  this  has  not  been  proved,  and,  on  the  other 
hand,  we  have  ample  evidence  that  widely  distributed  species  exist 
having  very  definite  morphological  and  biological  characters  which 
enable  us  to  recognize  them  wherever  found. 

It  has  generally  been  supposed  that  these  simple  vegetable  cells 
are  destitute  of  a  nucleus,  but  a  recent  author  (Frankel)  suggests 
the  probability  that  a  nucleus  may  exist,  although  it  has  not  been 
demonstrated.  This  suggestion  is  based  upon  the  fact  that  in  stain- 
ing bacteria  very  quickly  it  sometimes  happens  that  a  portion  of  the 
protoplasm  is  sharply  differentiated  by  taking  the  stain  more  deeply 
than  the  remaining  portion. 

Sjobring  in  1892  made  an  investigation  for  the  purpose  of 
ascertaining  the  structure  of  bacterial  cells.  Various  methods 
were  employed,  but  the  most  satisfactory  results  were  obtained  by 
fixing  with  nitric  acid,  with  or  without  alcohol,  and  without  pre- 


118  STRUCTURE,    MOTIONS,    REPRODUCTION. 

vious  drying  ;  the  preparations  were  then  stained  with  carbol-meth- 
ylene-blue  or  carbol-fuchsin  solution  ;  they  were  decolorized  with 
nitric  acid  and  examined  in  glycerin  or  in  water.  By  this  procedure 
the  author  named  was  able  to  demonstrate  two  kinds  of  corpuscles. 
One  of  these  may  be  seen  just  inside  the  cell  wall ;  it  stains  deeply 
with  the  carbol-fuchsin  solution.  The  other  lies  in  a  position  analo- 
gous to  that  occupied  by  the  nucleus  of  vegetable  cells  higher  in  the 
scale,  and  resembles  this  both  in  its  resting  condition  and  in  the 
process  of  indirect  division. 

In  his  addross  bofore  the  International  Medical  Congress  of  Ber- 
lin (1800)  Koch  says  : 

'  We  had  not  succeeded,  in  spite  of  the  constantly  improving 
methods  of  staining  and  in  spite  of  the  use  of  objectives  with  con- 
stantly increasing  angles  of  aperture,  in  learning  more  with  reference 
to  the  interior  structure  of  the  bacteria  than  was  shown  by  the  origi- 
nal methods  of  staining.  Only  very  recently  new  methods  of  stain- 
ing appear  to  give  us  further  information  upon  the  structure  of  the 
bacteria,  inasmuch  as  they  serve  to  differentiate  an  interior  portion 
of  the  protoplasm,  which  should  probably  be  regarded  as  a  nucleus, 
from  an  exterior  protoplasmic  envelope  from  which  is  given  off  the 
organ  of  locomotion,  the  flagellum." 

Although  usually  transparent,  the  protoplasm  sometimes  presents 
a  granular  appearance.  The  botanist  Van  Tieghem  claims  to  have 
found  chlorophyll  grains  in  some  water  bacteria  studied  by  him,  and 
in  the  genus  Beggiatoa  grains  of  sulphur  are  found  embedded  in  the 
protoplasm  of  certain  species. 

The  granules  in  bacterial  cells  which  may  be  demonstrated  by 
special  methods  of  staining  are  of  two  kinds :  metachromatic  gran- 
ules and  polar  granules.  The  former  lie  in  the  protoplasm,  and 
when  properly  stained  may  present  the  appearance  of  a  short  chain 
of  cocci  lying  in  the  bacterial  cell.  To  demonstrate  their  presence 
Ernst  recommends  the  use  of  Loffler's  solution  of  methylene  blue. 
This  is  placed  upon  a  cover-glass  preparation  and  heated  over  a 
flame  until  steam  begins  to  rise.  After  washing  in  water  the  cover 
glass  is  placed  for  a  minute  or  two  in  a  watery  solution  of  Bismarck 
brown.  This  shows  the  granules  stained  blue  and  the  surrounding 
protoplasm  brown.  The  polar  granules  are  often  seen  in  prepara- 
tions stained  in  the  usual  way  with  an  aniline  staining  solution. 
Some  observers  have  regarded  these  stained  granules  as  spores,  but 
this  has  not  been  demonstrated,  and  cultures  containing  them  show 
no  greater  resistance  to  heat  or  to  chemical  agents  than  that  estab- 
lished for  the  vegetative  cells  of  the  particular  species  in  which  they 
are  found.  It  seems  probable  that  the  matachromatic  granules  re- 
sult from  degenerative  changes  rather  than  that  they  are  reproduc- 
tive bodies. 


STRUCTURE,    MOTIONS,    REPRODUCTION.  119 

The  cell  membrane  in  certain  species  appears  to  be  very  flexible, 
as  may  be  seen  in  those  which  have  a  sinuous  motion.  It  is  not 
easily  recognized  under  the  microscope,  but  by  the  use  of  reagents 
which  cause  the  protoplasm  to  contract  may  be  demonstrated — e.g., 
by  iodine  solution.  Outside  of  the  true  cell  membrane  a  gelatinous 
envelope — so-called  capsule — is  sometimes  seen.  This  may  perhaps 
be,  as  claimed  by  some  authors,  nothing  more  than  a  jelly-like  thick- 
ening of  the  outer  layers  of  the  cell  wall.  This  jelly-like  material 
causes  the  cells  to  adhere  to  each  other,  forming  zooglcea  masses. 
In  some  cases  the  growth  upon  the  surface  of  a  culture  medium  is 
extremely  viscid,  and  may  be  drawn  out  into  long  threads  when 
touched  with  a  platinum  needle,  owing  to  the  gelatinous  intercellular 
substance  by  which  the  cells  are  surrounded. 

There  is  but  little  more  to  be  said  of  the  structure  of  these  minute 
organisms,  except  to  mention  the  fact  that  the  motile  species  are 
provided  with  slender,  whip-like  appendages  called  flagella.  The 
micrococci  in  general  are  not  endowed  with  the  power  of  executing 
spontaneous  movements,  and  they  are  not  provided  with  flagella. 
Bat  recently  two  motile  species  have  been  described,  and  in  one  of 
these — Micrococcus  agilis  of  Ali-Cohen — the  presence  of  flagella  has 
been  demonstrated. 

Many  of  the  bacilli  and  spirilla  are  actively  motile,  and  the  pre- 
sence of  flagella,  which  has  long  been  suspected,  has  recently  been 
demonstrated  for  a  considerable  number  of  species  by  Loffler  and 
others. 

It  must  be  remembered  that  the  molecular  movement  which  is 
common  to  all  minute  particles  suspended  in  a  fluid  is  a  vibratory 
motion  in  situ,  which  does  not  change  the  relative  position  of  the 
moving  particles.  This  so-called  Brownian  movement  has  frequently 
been  mistaken  for  a  vital  motion,  as  has  also  the  movement  due  to 
currents  in  the  liquid  in  which  non-motile  organisms  are  suspended. 
The  latter  is  to  be  distinguished  by  the  fact  that  the  microorganisms 
are  all  carried  in  one  direction.  This  movement  due  to  a  current,  in 
connection  with  the  vibratory  Brownian  movement,  is  very  deceptive, 
and  it  is  often  hard  for  a  beginner  in  bacteriological  study  to  con- 
vince himself  that  what  he  sees  is  not  a  vital  movement.  But  in 
true  vital  movements  we  have  progression  in  different  directions,  and 
the  individual  microorganisms  approach  and  pass  each  other,  often 
in  a  most  vigorous  and  active  manner,  passing  entirely  across  the 
field  of  view  or  changing  direction  in  an  abrupt  way.  Sometimes 
the  motion  is  slow  and  deliberate,  the  bacillus  progressing  with  a  to- 
and-fro  motion,  as  if  propelled  by  a  trailing  flagellum  ;  or  it  may  be 
serpentine  when  the  moving  filament  is  flexible ;  or  again  it  is 
a  darting  forward  motion  which  is  so  rapid  that  the  eye  can  scarcely 
follow  the  moving  body.  The  spirilla  have  a  rotary  movement  as 


120  STRUCTURE,    MOTIONS,    REPRODUCTION. 

well  as  a  progressive  one,  and  this  is  often  extremely  rapid.  Some- 
times bacilli  spin  around  with  a  rotatory  motion,  as  if  they  were  an- 
chored fast  to  a  fixed  point,  as  they  may  be  by  the  flagellum  being 
attached  to  the  slide  or  cover  glass.  Frequently,  in  a  pure  culture, 
the  individual  bacilli  may  be  seen  to  come  to  rest,  and,  after  an  inter- 
val of  repose,  to  dart  forward  again  in  the  most  active  way.  Or  we 
may  find,  on  examining  the  same  culture  at  different  times,  that 
upon  one  occasion  there  is  no  evidence  of  vital  movements,  and  on 
another  all  of  the  bacilli  are  actively  motile.  These  differences  de- 
pend upon  the  age  of  the  culture,  temperature  conditions,  etc. 

Reproduction  by  binary  division  is  common  to  all  of  the  bacte- 
ria, and  in  many  species  this  is  the  only  mode  of  reproduction  known. 
When  circumstances  are  favorable  for  rapid  multiplication  the  indi- 
vidual cells  grow  in  length,  and  a  constriction  occurs  in  the  middle 
transverse  to  the  long  diameter.  This  becomes  deeper,  and  after  a 
time  the  cell  is  completely  divided  into  two  equal  portions,  which 
again  divide  in  the  same  way.  Separation  may  be  complete,  or  the 
cells  may  remain  attached  to  each  other,  forming  chains  (strepto- 
cocci) or  articulated  filaments  (scheinfdden  of  the  Germans). 

The  bacilli  and  spirilla  divide  only  in  a  direction  transverse  to  the 
long  diameter  of  the  cells,  but  among  the  micrococci  division  may 
occur  either  in  one  direction,  forming  chains  ;  or  in  two  directions, 
forming  tetrads  ;  or  in  three  directions,  forming  "  packets  "of  eight 
or  more  elements.  The  staphylococci,  in  which  the  cells  do  not  re- 
main associated,  divide  indifferently  in  any  direction. 

The  rapidity  of  multiplication  by  binary  division  varies  greatly  in 
different  species,  and  in  the  same  species  depends  upon  conditions  re- 
lating to  the  culture  medium,  age  of  the  culture,  temperature,  etc. 
Under  favorable  conditions  bacilli  have  been  observed  to  divide  in 
twenty  minutes,  and  it  is  a  matter  of  common  laboratory  experience 
that  colonies  of  considerable  size  and  containing  millions  of  bacilli 
may  be  developed  from  a  single  cell  in  twenty-four  to  forty-eight 
hours.  A  simple  calculation  will  show  what  an  immense  number  of 
cells  may  be  produced  in  this  time  as  a  result  of  binary  division  oc- 
curring, for  example,  every  hour.  The  progeny  of  a  single  cell 
would  be  at  the  end  of  twenty-four  hours  16,777,220,  and  at  the  end 
of  forty-eight  hours  the  number  would  be  281,500,000,000. 

Some  of  the  earlier  observers  have  noted  the  presence  of  oval  or 
spherical  refractive  bodies  in  cultures  containing  bacilli ;  but  that 
these  were  reproductive  elements,  although  suspected,  was  not  de- . 
monstrated  until  a  comparatively  recent  date.  Pasteur  was  one  of 
the  first  to  point  out  the  fact  that  certain  bacteria  have  two  modes  of 
reproduction — by  fission  and  by  the  formation  of  endogenous  spores  ; 
but  the  first  careful  study  of  the  last-mentioned  method  was  made  by 


STRUCTURE,    MOTIONS,    REPRODUCTION.  121 

Koch  in  his  classical  stud}'  of  the  anthrax  bacillus  (1878),  and  by 
Cohn,  who  studied  the  formation  of  spores  in  Bacillus  subtilis. 

These  reproductive  bodies  serve  the  same  purpose  in  the  preserva- 
tion of  species  as  the  seeds  of  higher  plants.  They  resist  desiccation 
and  may  retain  their  vitality  for  months  or  years  until  circumstances 
are  favorable  to  their  development,  when,  under  the  influence  of  heat 
and  moisture,  they  reproduce  the  vegetative  form — bacillus  or  spiril- 
lum— with  all  of  its  biological  and  morphological  characters.  They 
are  composed  of  condensed  protoplasm  which  retains  the  vital  char- 
acters of  the  soft  protoplasm  of  the  mother  cell  from  which  it  has 
been  separated  ;  and  it  is  evident  that  whether  reproduction  occurs 
by  fission  or  by  the  formation  of  endogenous  spores,  the  protoplasm 
of  the  cells  in  a  pure  culture  of  any  microorganism  is  simply  a  sepa- 
rated portion  of  the  protoplasm  of  the  progenitors  of  these  cells. 

Some  of  the  bacilli  grow  out  into  long  filaments  before  the  forma- 
tion of  spores  occurs  ;  and  these  filaments  may  be  associated  in  bun- 
dles or  intertwined  in  irregular  masses.  At  first  the  protoplasm  of  the 


FIG.  75. 


filaments  is  homogeneous,  but  after  a  time  it  becomes  segmented, 
and  later  the  protoplasm  of  each  segment  becomes  condensed  into 
a  spherical  or  oval  refractive  body,  which  is  the  spore.  For  a  time 
these  are  retained  in  a  linear  position  by  the  cell  membrane  of  the 
filament  (Fig.  75,  a),  but  this  is  after  a  while  dissolved  or  broken 
up  and  the  spores  are  set  free.  In  liquid  cultures  they  sink  to  the 
bottom  as  a  pulverulent  precipitate,  and  upon  the  surface  of  a  solid 
medium  they  form  a  layer  which  is  usually  of  a  white  or  yellowish- 
white  color,  and  which,  when  examined  under  the  microscope,  in  old 
cultures  is  found  to  consist  almost  entirely  of  shining  spherical  or 
oval  bodies  which  do  not  stain,  by  the  ordinary  methods,  with  the 
aniline  colors.  While  many  of  the  bacilli  during  the  stage  of  spore 
formation  grow  out  into  long  filaments,  others  do  not,  and  one  or 
more  spores  make  their  appearance  in  rods  of  the  ordinary  length 
which  characterizes  the  species.  These  may  be  located  in  the  centre 
of  the  rod  or  at  one  extremity  (Fig.  75,  b).  It  sometimes  occurs 
that  when  a  single  central  spore  is  formed  the  rod  becomes  very 
much  enlarged  in  its  central  portion,  assuming  a  spindle  shape  (Fig. 


122  STRUCTURE,    MOTIONS,    REPRODUCTION. 

75  c) ;  or  one  extremity  may  be  enlarged,  producing  forms  such  as 
are  shown  in  Fig.  75,  d.  Some  of  the  smaller  spherical  spores  meas- 
ure less  than  0. 5  //.  in  diameter,  but  they  are,  for  the  most  part,  oval 
bodies  having  a  short  diameter  of  0.5  to  1  //-  and  a  long  diameter  of 
one  to  two  /^,  or  even  more.  They  are  enveloped  in  a  cellular  en- 
velope which,  according  to  some  observers,  consists  of  two  layers— 
an  exosporium  and  an  endosporium. 

The  mode  of  spore  formation  shown  in  Fig.  75,  c  and  d,  has  been 
adopted  by  some  authors  as  a  generic  character.  When  the  spores 
are  located  in  the  central  part  of  the  rods,  giving  rise  to  a  spindle- 
shaped  body,  as  at  c,  the  bacilli  are  assigned  to  the  genus  Clos- 
tridium;  when  located  at  one  end,  as  at  d,  the  bacilli  are  shaped 
like  a  drumstick,  and  this  mode  of  spore  formation  is  used  as  the 
distinguishing  characteristic  of  the  genus  Plectridium.  Hueppe 
groups  all  rod-shaped  bacteria  which  form  endospores  under  the 
generic  name  Bacillus,  with  three  sub-genera :  Bacillus,  straight 
rods;  Clostridium,  spindle-shaped  rods;  Plectridium,  drumstick- 
shaped  rods. 

The  germination  of  spores  has  been  studied  by  Prazmowski, 
Brefeld,  and  others.  The  process  is  as  follows  :  By  the  absorption 
of  water  they  become  swollen  and  pale,  losing  their  shining,  refrac- 
tive appearance.  Later  a  little  protuberance  is  seen  upon  one  side 
or  at  one  extremity  of  the  spore,  and  this  rapidly  grows  out  to  form 
a  rod  which  consists  of  soft-growing  protoplasm  enveloped  in  a 
membrane  which  is  formed  of  the  endosporium  or  inner  layer  of  the 
cellular  envelope  of  the  spore.  The  outer  envelope,  or  exosporium, 
is  cast  off  and  may  be  seen  in  the  vicinity  of  the  newly  formed  rod 
(Fig.  76).  Sometimes  the  vegetative  cell  emerges  from  one  extrem- 


ity of  the  oval  spore,  as  shown  at  a,  Fig.  76,  and  in  other  species  the 
exosporium  is  ruptured  and  the  bacillus  emerges  from  the  side,  as 
seen  at  b. 

The  considerable  resistance  of  these  endogenous  spores  to  desic- 
cation, to  heat,  and  to  various  chemical  agents  is  an  important  fact 
both  from  a  biological  and  from  a  hygienic  point  of  view,  and  will 
be  fully  considered  in  a  subsequent  chapter.  The  fact  that  certain 
bacilli  and  spirilla  do  not  withstand  a  temperature  of  80°  to  90°  C., 
which  does  not  destroy  the  vitality  of  known  spores,  leads  to  the  in- 


STRUCTURE,    MOTIONS,    REPRODUCTION. 

ference  that  they  do  not  form  similar  reproductive  bodies.  But  re- 
productive elements  of  a  different  kind  are  described  by  some  botan- 
ists as  being  produced  during  the  development  of  these  bacteria,  and 
also  of  the  micrococci.  These  are  the  so-called  arthroxpores.  In 
the  process  of  binary  division  certain  cells  in  a  chain  ma}7  be  observed 
to  be  somewhat  larger  than  others  and  to  refract  light  more  strongly. 
The  same  may  be  true  of  certain  cells  in  a  culture  in  which  the  ele- 
ments are  not  united  in  chains.  These  cells  are  believed  by  De  Bary 
and  others  to  have  greater  resisting  power  to  desiccation  than  the  re- 
maining cells  in  the  culture,  and  to  serve  the  purpose  of  reproductive 
elements. 

Hueppe  groups  all  rod-shaped  bacteria  which  do  not  form  endo- 
spores  under  the  generic  name  Arthrobacterium.  This  author  be- 
lieves that,  as  a  rule,  bacilli  which  do  not  form  endospores  under 
certain  circumstances  produce  more  resistant  cells  which  "take 
charge  of  the  perpetuation  of  the  species  under  the  guise  of  a  resting 
stage  or  spore."  According  to  Hueppe,  true  arthrospores  are  spheri- 
cal in  form. 

It  has  generally  been  supposed  that  spore  formation  is  most  likely 
to  occur  when  the  pabulum  for  supporting  the  growth  of  the  vegeta- 
tive form  is  nearly  exhausted.  But,  as  pointed  out  by  Frankel,  facts 
do  not  support  this  view,  as  many  species  form  spores  when  condi- 
tions are  most  favorable  for  a  continued  development.  An  abundant 
supply  of  oxygen  favors  the  formation  of  spores  in  aerobic  species, 
and,  in  some  instances  at  least,  the  temperature  has  an  important  in- 
fluence upon  spore  formation.  Thus  the  anthrax  bacillus  does  not 
form  spores  at  temperatures  below  20°  C.  or  above  42°  C. 

The  very  interesting  fact  has  been  demonstrated  by  Lehman  and 
by  Behring  that  a  species  which  usually  forms  spores  may  be  so 
modified  by  certain  influences  that  it  is  no  longer  capable  of  spore 
production,  and  that  such  an  asporogenous  variety  may  be  cultivated 
for  an  indefinite  time  without  showing  any  return  to  the  stage  of 
spore  formation.  This  was  effected  in  Behring's  experiments  by 
cultivating  the  anthrax  bacillus  in  a  medium  containing  some  agent 
detrimental  to  the  vitality  of  the  vegetative  cells,  but  not  in  suffi- 
cient quantity  to  restrain  their  development. 

The  pseudo-branching  of  the  filaments  in  the  genus  Cladothrix 
has  been  referred  to  in  the  chapter  on  Morphology.  Recent  re- 
searches show  that  other  bacteria  heretofore  included  in  the  -genus 
Bacillus  may  also  present  branching  forms.  This  is  especially  true 
of  the  tubercle  bacillus,  which  when  obtained  from  cases  of  fowl 
tuberculosis  not  infrequently  exhibits  a  sort  of  branching.  Hueppe 
and  Fischel  have  also  demonstrated  the  presence  of  branching  forms 
of  the  bacillus  of  mammalian  tuberculosis,  and  as  a  result  of  his  ob- 


124  STRUCTURE,    MOTIONS,    REPRODUCTION. 

servations  Hueppe  has  "arrived  at  the  definite  opinion  that  the 
tubercle  bacillus  is  the  parasitic  growth- form  of  a  pleomorphic  mould, 
and  is  not  a  true  bacterium  at  all."  Metschnikoff  has  reported  his 
observations  of  branching  forms  of  the  cholera  spirillum,  Frankel  of 
the  diphtheria  bacillus,  and  Semmer  of  the  bacillus  of  glanders,  but 
whether  these  are  examples  of  pseudo-branching,  such  as  occurs  in  the 
genus  Cladothrix,  or  a  veritable  dichotomous  growth  such  as  occurs 
in  the  mould  fungi,  has  not  been  definitely  determined. 

The  chemical  composition  of  the  bacterial  cells  has  been  inves- 
tigated by  Nencki,  Brieger,  and  others.  Putrefactive  bacteria  culti- 
vated in  a  two-per-cent  solution  of  gelatin,  and  which  produced  an 
abundant  intercellular  substance  connecting  the  cells  in  zoogloea 
masses,  were  found  by  Xeiicki  to  have  the  following  composition  : 
Water,  84.26  per  cent;  solids,  5.74  per  cent,  consisting  of  albumin 
87.46  per  cent,  fat  6.41,  ash  3.04,  undetermined  remnant  3.09. 
The  albuminous  substance,  according  to  Nencki,  is  not  precipitated 
by  alcohol,  and  differs  in  its  chemical  composition  from  other  known 
substances  of  this  class.  He  calls  it  wykoprotein  and  gives  the  fol- 
lowing as  its  chemical  composition  :  C,  52.32  percent;  H,  7.55  per 
cent ;  N",  14. 75  per  cent.  It  contains  no  sulphur  and  no  phosphorus. 
The  spores  of  the  anthrax  bacillus,  according  to  Nencki,  do  not  con- 
tain mykoprotein,  but  a  peculiar  albuminous  substance  which  he 
calls  anthrax-protein.  Brieger  analyzed  a  gelatin  culture  of  Fried- 
lander's  bacillus,  with  the  following  result  :  Water,  84.2  per  cent ; 
solids,  5.8  per  cent,  containing  1.74  per  cent  of  fats.  After  removal 
of  the  fat  the  solids  gave  an  ash  of  30.13  per  cent  ;  this  contains  cal- 
cium phosphate,  magnesium  phosphate,  sodium  sulphate,  and  sodium 
chloride.  The  amount  of  nitrogen  in  the  dried  substance  after  re- 
moval of  the  fat  was  9. 75. 


II. 

CONDITIONS  OF  GROWTH. 

BACTERIA  only  grow  in  presence  of  moisture,  under  certain  condi- 
tions of  temperature,  and  when  supplied  with  suitable  pabulum.  As 
they  'do  not  contain  chlorophyll,  they  cannot  assimilate  carbon  diox- 
ide, and  light  is  not  favorable  to  their  development. 

The  aerobic  species  obtain  oxygen  from  the  air  and  cannot  grow 
unless  supplied  with  it.  The  anaerobic  species,  on  the  other  hand, 
will  not  grow  in  the  presence  of  oxygen,  and  must  obtain  this  ele- 
ment, as  they  do  carbon  and  nitrogen,  from  the  organic  material 
which  serves  them  as  food. 

As  a  class  the  bacteria  are  supplied  with  nutriment  by  the  higher 
plants  and  animals,  the  dead  tissues  of  which  they  appropriate,  and 
which  it  is  their  function  to  decompose,  releasing  the  organic  ele- 
ments as  simple  compounds  which  may  again  be  assimilated  by  the 
chlorophyll-producing  plants. 

Water  is  essential  for  the  development  of  bacteria,  and  many  spe- 
cies have  their  normal  habitat  in  the  waters  of  the  ocean,  of  lajses, 
and  of  running  streams  ;  others  thrive  upon  damp  surfaces  or  in  the 
interior  of  moist  masses  of  organic  material.  Many  species  grow  in- 
differently either  in  salt  or  fresh  water,  but  it  is  probable  that  cer- 
tain species  will  be  found  peculiar  to  the  waters  of  the  ocean.  Some 
of  the  water  bacteria  multiply  in  the  presence  of  an  exceedingly 
minute  amount  of  organic  pabulum,  or  even  in  distilled  water.  This 
is  shown  by  the  experiments  of  Bolton  and  others.  The  author 
named  tested  two  species  of  water  bacteria  (Micrococcus  aquatilis 
and  Bacillus  erythrosporus)  in  the  following  manner :  Ten  cubic 
centimetres  of  distilled  water  in  a  test  tube  were  infected  with  a  small 
quantity  of  a  culture  of  one  of  these  microorganisms.  A  drop  from 
this  tube  was  transferred'  to  the  same  quantity  of  distilled  water  in 
a  second  tube,  and  from  this  to  a  third.  The  number  of  bacteria  in 
this  tube  No.  3  was  now  ascertained  by  counting,  and  it  was  put 
aside  for  two  or  three  days,  at  the  end  of  which  time  the  number  was 
again  estimated  by  counting.  In  every  case  there  was  an  enormous 
increase  in  the  number  of  bacteria.  In  order  to  be  sure  that  the  dis- 


126  CONDITIONS    OF   GROWTH. 

tilled  water  was  pure,  it  was  distilled  a  second  time  in  a  clean  glass 
retort,  but  the  result  was  the  same.  Bolton  remarks,  with  reference 
to  these  results:  "If  we  seek  to  explain  this  remarkable  fact  we 
must  remember,  in  the  first  place,  what  an  extremely  small  abso- 
lute mass  is  represented  by  an  enormous  number  of  bacteria,  and 
what  a  minute  amount  of  material  is  required  for  the  formation  of 
this  mass.  In  ten  cubic  centimetres  of  distilled  water,  in  the  experi- 
ment last  referred  to,  there  were  about  twenty  million  bacteria  (two 
million  per  cubic  centimetre).  If  we  estimate  the  diameter  of  each 
at  one  yu,  with  a  specific  weight  of  1,  the  absolute  weight  would 
be  for  the  entire  number  one-one-hundredth  of  a  milligramme— 
that  is  to  say,  a  quantity  which  cannot  be  determined  by  any  of  our 
methods  of  weighing." 

Bolton  supposes  that  the  small  amount  of  organic  pabulum  re- 
quired fell  into  the  water  in  the  shape  of  dust,  or  was  attached  to  the 
walls  of  the  test  tube  in  spite  of  all  the  precautions  taken. 

Nitrogen  is  chiefly  obtained  from  albuminoid  substances,  but 
Pasteur  has  shown  that  it  may  also  be  obtained  from  ammonia. 
This  is  shown  by  cultivating  bacteria  in  a  medium  containing  an 
ammonia  salt,  as  in  the  following  :  * 

PASTEUR'S  SOLUTION. 

Distilled  water,               ......  100 

Cane  sugar,              ......  10 

Tartrate  of  ammonia,    ......  1 

Ashes  of  one  gramme  of  yeast,     ....  0.075 

CORN'S  SOLUTION. 

'Distilled  water,  ......  100 

Tartrate  of  ammonia,  .....  1 

Ashes  of  yeast,  .......  1 

Many  bacteria  multiply  abundantly  in  these  solutions. 

Carbon  is  obtained  from  the  various  organic  substances  contain- 
ing it ;  among  others,  from  starch,  sugars,  glycerin,  organic  acids 
and  their  salts,  etc. 

Temperature. — There  are  certain  limits  of  temperature  within 
which  development  may  take  place,  but  these  dift'er  greatly  with 
different  species.  As  a  rule,  growth  is  arrested  when  the  tempera- 
ture falls  below  10°  C.  (50°  F.),  but  some  species  multiply  at  a  still 
lower  temperature.  Thus  Bolton  observed  a  very  decided  increase 
in  certain  water  bacteria  kept  in  an  ice  chest  at  6°  C.,  and  other  ob- 
servers have  witnessed  development  at  the  freezing  temperature. 

Most  saprophytic  bacteria  grow  within  rather  wide  temperature 
limits,  but  the  rapidity  of  development  is  greatest  at  a  certain  favor- 
able temperature,  which  is  usually  between  25°  and  30°  C.  The 


CONDITIONS    OF   GROWTH.  127 

parasitic  species  have  a  more  restricted  range,  which  approaches  the 
normal  temperature  of  the  animals  in  which  they  habitually  de- 
velop. At  40°  C.  (104°  F.)  growth,  as  a  rule,  ceases,  hut  there  are 
some  notable  exceptions  to  this  rule. 

Miquel  some  years  ago  found  a  bacillus  in  the  water  of  the  Seine 
which  grew  at  a  temperature  of  69°  to  70°  C. ;  Van  Tieghem  reports 
having  observed  species  in  thermal  waters  capable  of  growth  at  a 
still  higher  temperature  (74°  C.)  ;  and  Globig  has  more  recently  ob- 
tained from  garden  earth  several  species  which  multiplied  at  G5°  C. 
Some  of  the  species  found  by  the  last-named  observer  were  even 
found  to  require  a  temperature  of  about  60°  for  their  development ; 
and  yet  this  temperature  is  quickly  fatal  to  a  large  number  of  the 
best  known  species. 

Loiv  temperatures,  while  arresting  the  growth  of  bacteria,  do  not 
destroy  their  vitality.  This  has  been  demonstrated  by  numerous  ex- 
periments, in  which  they  have  been  exposed  for  hours  in  a  refrigerat- 
ing mixture  at  —18°  C.  Frisch  has  even  subjected  them  to  a  tempe- 
rature of  —  87°  C.  by  the  evaporation  of  liquid  carbon  dioxide,  and 
found  that  they  still  grew  when  placed  in  favorable  conditions. 

Parasitism. — The  strict  parasites  grow  only  in  the  bodies  of  liv- 
ing animals,  or  in  artificial  media  kept  at  a  suitable  temperature. 
As  examples  we  may  mention  the  bacillus  of  tuberculosis,  the  bacil- 
lus of  leprosy,  the  micrococcus  of  gonorrhoea,  the  spirillum  of  re- 
lapsing fever.  There  is  also  a  large  class  of  facultative  para- 
sites which,  when  introduced  into  the  body  of  a  susceptible  animal, 
multiply  in  it,  and  may  continue  to  live  as  parasites  so  long  as  they 
are  transferred  from  one  animal  to  another,  but  which  are  also  able 
to  live  as  saprophytes  independently  of  a  living  host.  To  this  class 
belong  the  pus  cocci,  the  bacillus  of  typhoid  fever,  the  spirillum  of 
cholera,  and  many  others. 

It  seems  extremely  probable  that  the  strict  parasites  were  at  one 
time  capable  of  living  a  saprophytic  existence,  and  that  their  restric- 
tion to  a  parasitic  mode  of  life  has  been  effected  in  course  of  time  in 
accordance  with  the  laws  of  natural  selection.  This  view  is  sup- 
ported by  the  fact  that  the  tubercle  bacillus,  which  has  been  regarded 
as  a  strict  parasite,  which  can  only  be  cultivated  artificially  under 
very  special  conditions,  has  been  shown  co  be  capable  of  modification  in 
this  regard  to  such  an  extent  that  when  cultivated  for  a  time  in  a  favor- 
able medium — bouillon  with  five  per  cent  of  glycerin — it  will  even  grow 
in  ordinary  bouillon  made  from  the  flesh  of  a  calf  or  a  fowl  (Roux). 

Reaction  of  Medium. — Some  bacteria  grow  readily  in  a  medium 
having  an  acid  reaction,  while  the  slightest  trace  of  acidity  prevents 
the  development  of  others.  As  a  rule,  the  pathogenic  species  require 
a  neutral  or  slightly  alkaline  culture  medium. 


128  CONDITIONS   OF   GROWTH. 

While  many  species  grow  in  various  media  and  under  various 
conditions  of  temperature,  etc.,  others  are  greatly  restricted  in  this 
regard  ;  thus  Bumm  only  succeeded  in  cultivating  the  gonococcus 
upon  human  blood  serum,  and  even  upon  this  was  not  able  to 
carry  it  through  a  series  of  successive  cultures.  It  is  very  probable 
that  certain  species  can  only  grow  in  association  with  others  which 
elaborate  products  necessary  for  their  development. 

Substances  favorable  for  the  growth  of  a  particular  species  may 
restrain  its  development  if  present  in  too  large  an  amount.  Thus 
the  phosphorescent  bacilli  multiply  abundantly  in  a  nutrient  solution 
containing  2. 5  per  cent  of  sodium  chloride  ;  but  this  amount  would 
restrain  the  development  of  some  other  species,  and  a  considerable 
increase  in  the  quantity  of  salt  prevents  the  growth  of  all  microor- 
ganisms. In  the  same  way  the  addition  of  two  per  cent  of  glucose 
to  culture  solutions  is  favorable  for  the  development  of  certain  spe- 
cies, and  especially  for  the  anaerobic  bacteria  ;  but  a  concentrated 
solution  of  the  same  substance  prevents  the  growth  of  all  bacteria. 

The  influence  of  one  species  upon  the  groivth  of  another  has 
been  studied  by  various  bacteriologists,  and  especially  by  Sirotinin 
and  by  Freudenreich.  When  several  species  are  associated  in  the 
same  culture  one  may  take  the  precedence  and  the  others  may  de- 
velop later  ;  or  two  or  more  species  may  develop  at  the  same  time  ; 
or  the  growth  of  one  species  may  prevent  the  development  of  an- 
other, either  (a)  by  exhausting  the  pabulum  necessary  for  its  growth 
or  (6)  by  producing  substances  which  inhibit  the  development  of  an- 
other species  or  destroy  its  vitality. 

Freudenreich  found,  as  a  result  of  his  numerous  experiments, 
that  the  following  species  cause  a  change  in  bouillon  which  renders 
it  unfit  for  the  growth  of  other  species :  Bacillus  pyocyaneus,  Bacil- 
lus cyanogenus,  Bacterium  phosphorescens,  Bacillus  prodigiosus,  Spi- 
rillum cholerse  Asiaticse.  The  following  species  do  not  cause  such  a 
change  in  bouillon  as  to  render  it  unfit  for  the  growth  of  other  spe- 
cies :  Bacillus  typhi  abdominalis,  Bacillus  aiithracis,  Bacillus  septi- 
ca3mise  hsemorrhagica3,  Spirillum  tyrogenum.  The  following  have  a 
decided  antagonism  :  Bacillus  pyogenes  fcetidus  prevents  the  growth 
of  Spirillum  cholerse  Asiaticse  ;  Micrococcus  roseus  prevents  the 
growth  of  Micrococcus  tetragenus.  The  cholera  spirillum  will  not 
grow  in  sterilized  cultures  of  Bacillus  pyocyaneus,  or  in  bouillon 
which  has  served  for  a  previous  culture  of  the  same  microorganism 
(Kitasato).  Other  bacteria  which  fail  to  grow  in  bouillon  which 
has  already  served  for  the  cultivation  of  the  same  species  are  Bacil- 
lus typhi  abdominalis,  Bacillus  cyanogenus,  Bacillus  prodigiosus, 
Micrococcus  roseus,  etc.  (Freudenreich). 


III. 

MODIFICATIONS  OF  BIOLOGICAL  CHARACTERS. 

WE  have  already  referred  to  the  production  of  an  asporogenous 
variety  of  the  anthrax  bacillus.  This  was  effected  by  Behring  by 
cultivation  in  media  containing  small  amounts  of  hydrochloric  acid, 
caustic  soda,  methyl  violet,  malachite  green,  and  various  other 
agents.  This  is  only  one  of  many  instances  of  a  change  in  biologi- 
cal characters  due  to  changed  conditions  of  environment.  We  have 
abundant  experimental  evidence  that  growth  may  occur  under  ad- 
verse conditions  when  the  species  is  gradually  habituated  to  these 
conditions.  Thus  the  temperature  limitations  may  be  passed  by  suc- 
cessive cultivations  at  temperatures  approaching  these  limits,  and 
bacteria  may  grow  in  the  presence  of  agents  which  in  a  given  pro- 
portion have  a  complete  restraining  influence  upon  their  develop- 
ment. For  example,  in  the  experiments  of  Kossiakoff,  published  in 
the  Annales  of  the  Pasteur  Institute  (vol.  i.),  it  was  found  that  the 
several  species  tested  all  became  habituated  to  the  presence  of  anti- 
septic agents  in  proportions  which  at  first  completely  restrained 
their  growth. 

This  modification  of  biological  characters  is  well  shown  in  the 
case  of  the  chromogenic  bacteria,  some  of  which  only  form  pig- 
ment under  exceptionally  favorable  conditions  of  growth.  It  has 
been  shown  by  several  observers  that  iion-chromogenic  varieties 
of  some  of  the  best  known  chromogenic  species  may  be  produced 
by  special  methods  of  cultivation.  Thus  Wasserzug  obtained  a 
non-chromogenic  variety  of  the  bacillus  of  green  pus  (Bacillus 
pyocyaneus)  by  the  action  of  time  added  to  that  of  antiseptics.  He 
says:  "  These  two  actions  combined  have  permitted  me  to  obtain 
cultures  which  remained  without  color  in  a  durable  way,  and  in 
which,  consequently,  the  chromogenic  function  was  abolished  by 
heredity."  In  the  case  of  a  chromogenic  bacillus  obtained  by  the 
writer  in  Havana  (my  Bacillus  Havaniensis),  a  non-chromogenic  vari- 
ety was  obtained  from  a  culture  on  nutrient  agar  which  had  been  kept 
in  a  hermetically  sealed  glass  tube  for  about  a  year.  The  variety 
preserved  the  morphological  characters  of  the  original  stock,  but,  al- 
0 


130  MODIFICATIONS   OF   BIOLOGICAL   CHARACTERS. 

though  carried  through  successive  cultures  for  a  considerable  period, 
did  not  regain  its  power  to  produce  the  brilliant  carmine  color  which 
is  the  most  striking  character  of  the  species.  Katz,  in  cultivating 
the  phosphorescent  bacilli  isolated  by  him  from  sea  water  at  New 
South  Wales,  found  that,  after  being  propagated  for  some  time  in 
artificial  media,  their  power  to  give  off  a  phosphorescent  light  was 
diminished  or  temporarily  lost.  He  also  found  that  two  species 
which  when  first  cultivated  did  not  liquefy  gelatin,  subsequently, 
after  a  year,  caused  liquefaction  of  the  usual  gelatin  medium. 

Modification  shoivn  in  Cultures. — When  bacteria  have  been 
subjected  to  the  action  of  heat  or  chemical  agents,  without  having 
their  vitality  completely  destroyed,  they  often  show  diminished  vigor 
of  growth.  Cultures  which  would  ordinarily  show  an  abundant  de- 
velopment within  twenty-four  hours  may  not  commence  to  grow  for 
several  days.  For  this  reason,  in  disinfection  experiments,  it  is  neces- 
sary to  test  the  question  of  destruction  of  vitality  by  leaving  the  cul- 
tures for  a  week  or  -more  under  favorable  conditions  as  to  tempera- 
ture. In  plate  cultures  or  Esmarch  roll  tubes  a  few  colonies  may 
develop  in  this  tardy  way,  showing  that  there  was  a  difference  in  the 
vital  resisting  power  of  the  individual  cells,  some  having  survived 
while  the  majority  were  killed.  This  is  well  illustrated  by  Abbott's 
experiments  upon  the  germicidal  action  of  mercuric  chloride  as  tested 
upon  Staphylococcus  pyogenes  aureus.  Irregularities  in  the  results  in 
experiments  in  which  the  conditions  were  identical  having  been  no- 
ticed, Abbott  inferred  that  this  was  due  to  a  difference  in  the  resist- 
ing power  of  individual  cocci  (arthrospores  ?).  By  making  cul- 
tures from  colonies  which  developed  from  these  more  resistant  cocci, 
and  again  exposing  the  micrococci  in  these  cultures  to  mercuric  chlo- 
ride in  the  proportion  of  1:1,000  for  a  longer  time  and  making  new 
cultures  from  the  surviving  cocci,  and  so  on,  Abbott  obtained  cultures 
in  which  a  majority  of  the  cells  survived  exposure  to  a  solution  of  the 
strength  mentioned  for  ten  to  twenty  minutes,  whereas  in  his  original 
culture  most  of  the  cocci  were  killed  by  this  solution  in  five  minutes. 

These  changes  in  vital  resisting  power  enable  us  to  comprehend 
other  modifications  which  can  only  be  detected  by  chemical  or  bio- 
logical reactions.  Thus  the  reducing  power  for  various  substances 
may  be  modified  by  changes  in  the  conditions  of  environment.  And 
among  the  pathogenic  bacteria  changes  of  a  more  or  less  permanent 
nature  may  be  induced,  which  are  shown  by  a  modified  degree  of 
virulence  when  injected  into  susceptible  animals. 

Attenuation  of  Virulence  may  be  effected  by  several  methods, 
all  of  which  depend  upon  subjecting  the  cultures  to  prejudicial  in- 
fluences of  one  kind  or  another. 

Pasteur  first  announced,  in  1880,  that  the  microbe  of  fowl  cholera 


MODIFICATIONS   OF   BIOLOGICAL   CHARACTERS.  131 

could  be  modified  by  special  treatment  in  such  a  manner  that  it  no 
longer  produced  a  fatal  form  of  the  disease.  He  found  that  the  viru- 
lence was  greatest  when  cultures  were  made  from  fowls  which  had 
died  from  a  chronic  form  of  the  disease,  and  that  this  virulence  was 
not  lost  by  successive  cultivations  in  chicken  bouillon,  repeated  at 
short  intervals.  But  when  an  interval  of  more  than  two  months 
was  allowed  to  elapse  without  renewing  the  cultures,  the  virulenco 
was  diminished  and  fewer  deaths  occurred  in  fowls  inoculated  with 
such  cultures.  This  diminution  of  virulence  became  more  marked 
in  proportion  to  the  length  of  time  during  which  a  culture  solution 
containing  the  microbe  remained  exposed  to  the  action  of  the  atmo- 
sphere, and  at  last  all  virulence  was  lost  as  a  result  of  the  death  of 
the  pathogenic  microorganism.  When  the  virus  was  preserved  in 
hermetically  sealed  tubes  it  did  not  undergo  this  modification,  but  re- 
tained its  full  virulence  for  many  months.  According  to  Pasteur, 
the  various  degrees  of  modification  of  virulence  resulting  from  pro- 
longed exposure  to  the  air  may  be  preserved  in  successive  cultures 
made  at  short  intervals.  Subsequent  experiments  with  cultures  of 
the  anthrax  bacillus  gave  similar  results  and  enabled  him  to  produce 
an  "  attenuated  virus  "  for  his  protective  inoculations. 

In  the  case  of  the  anthrax  bacillus  it  was-  found  that  the  spores 
retain  their  full  virulence  for  years,  and  that  the  production  of  an  at- 
tenuated virus  required  the  exclusion  of  these  reproductive  elements. 
Cultivations  were  consequently  made  at  a  temperature  of  42°  to  4o° 
C.,  at  which  point  this  bacillus  is  incapable  of  producing  spores. 
Cultivation  at  this  temperature  for  eight  days  gave  an  attenuated 
virus  suitable  for  use  in  protective  inoculations. 

Attenuation  by  Heat. — Toussaint  has  shown  that  a  similar  modi- 
fication of  virulence  may  be  produced  by  exposure  for  a  short  time 
to  a  temperature  a  little  below  that  which  destroys  the  vitality  of  the 
pathogenic  organism.  This  is  best  accomplished,  according  to  Chau- 
veau,  in  the  case  of  the  bacillus  of  anthrax,  by  exposure  for  eighteen 
minutes  to  a  temperature  of  50°  C.  Exposure  to  this  temperature  for 
twenty  minutes  is  said  to  completely  destroy  the  vitality  of  the  bacillus. 

Attenuation-  IHJ  Antiseptic  Agents. — The  writer,  in  -1880,  ob- 
tained evidence  that  attenuation  of  virulence  may  result  from  ex- 
posure to  the  action  of  antiseptic  agents.  In  a  series  of  experiments 
made  to  determine  the  comparative  value  of  disinfectants,  the  blood 
of  a  rabbit  recently  dead  from  a  form  of  septica3inia  induced  by  the 
subcutaneous  injection  of  my  own  saliva,  and  due  to  the  presence  of 
a  micrococcus  (Micrococcus  pneumonia  crouposa3),  was  subjected  to 
the  action  of  various  chemical  agents,  a-nd  subsequently  injected 
into  a  rabbit  to  test  the  destruction  of  virulence.  In  the  published 
report  of  these  experiments  the  following  statement  is  made  : 


132  MODIFICATIONS    OF   BIOLOGICAL   CHARACTERS. 

'  The  most  important  source  of  error,  however,  and  one  which 
must  be  kept  in  view  in  future  experiments,  is  the  fact  that  a  pro- 
tective influence  has  been  shown  to  result  from  the  injection  of  virus 
the  virulence  of  which  has  been  modified,  without  being  entirely  de- 
stroyed, by  the  agent  used  as  a  disinfectant. " 

"Sodium  hyposulphite  and  alcohol  were  the  chemical  reagents 
which  produced  the  result  noted  in  these  experiments  ;  but  it  seems 
probable  that  a  variety  of  antiseptic  substances  will  be  found  to  be 
equally  effective  when  used  in  proper  proportion.  Subsequent  ex- 
periments have  shown  that  neither  of  these  agents  is  capable  of  de- 
stroying the  vitality  of  the  septic  micrococcus  in  the  proportion  used 
(one  per  cent  of  sodium  hyposulphite  or  one  part  of  ninety -five-per- 
cent alcohol  to  three  parts  of  virus),  and  that  both  have  a  restraining 
influence  upon  the  development  of  this  organism  in  culture  fluids. " 

Cultivation  in'  the  Blood  of  an  Immune  Animal. — It  has 
been  shown  by  the  experiments  of  Ogata  and  Jasuhara  that  when 
the  anthrax  bacillus  is  cultivated  in  the  blood  of  an  immune  animal, 
such  as  the  dog  or  the  white  rat,  its  pathogenic  power  is  modified 
so  that  it  no  longer  kills  susceptible  animals  and  may  be  used  as  a 
vaccine. 

Pasteur  had  previously  shown  (1882)  that  the  virus  of  rouget  can 
be  attenuated  by  passing  it  through  rabbits. 

Recovery  of  Virulence. — Pasteur  has  shown  that  when  the  viru- 
lence of  a  pathogenic  organism  has  been  modified  it  may  be  re- 
stored by  successive  inoculations  into  susceptible  animals.  Thus  in 
the  case  of  the  anthrax  bacillus  a  culture  which  would  not  kill  an 
adult  guinea-pig  may  be  inoculated  into  a  very  young  animal  of  the 
same  species  with  a  fatal  result ;  and  by  inoculating  the  blood  of 
this  animal  into  another,  and  so  on,  the  original  virulence  may  be 
restored,  so  that  a  culture  is  obtained  which  will  kill  a  sheep.  In. 
the  same  way  the  attenuated  virus  of  fowl  cholera  may  be  restored 
to  full  vigor  by  inoculating  a  small  bird — sparrow  or  canary — to 
which  it  is  fatal.  After  several  successive  inoculations  the  virus 
resumes  its  original  activity. 

In  general,  pathogenic  virulence  is  increased  by  successive  inocu- 
lations into  susceptible  animals,  and  diminished  by  cultivation  in  arti- 
ficial media  under  unfavorable  conditions.  Thus  various  pathogenic 
bacteria  which  have  been  cultivated  in  laboratories  for  a  length  of 
time  are  likely  to  disappoint  the  student  if  he  makes  inoculation  ex- 
periments for  the  purpose  of  demonstrating  their  specific  action  as 
described  in  text  books. 

1  Quoted  from  "  Bacteria,"  pages  207,  208,  written  in  1883. 


IT. 
PRODUCTS  OF  VITAL  ACTIVITY. 

ALL  living  cells,  animal  or  vegetable,  while  in  active  growth, 
appropriate  certain  elements  for  their  nutrition  from  the  pabulum 
with  which  they  are  supplied,  and  at  the  same  time  excrete  certain 
products  which,  in  some  cases  at  least,  it  is  their  special  function  to 
produce.  In  the  higher  plants  and  animals  specialized  cells  excrete 
substances  which  are  injurious  to  the  economy  of  the  individual, 
and  secrete  substances  which  are  required  to  maintain  its  existence. 
As  an  example  in  animals  we  may  mention  the  excretion  of  urea  by 
the  epithelium  of  the  kidneys,  the  retention  of  which  is  fatal  to  the 
individual,  and  the  gastric  secretion  which  is  essential  for  its  con- 
tinued existence.  Among  the-  higher  plants  we  have  an  immense 
variety  of  substances  formed  in  the  cell  laboratories,  some  of  which 
are  evidently  useful  for  the  preservation  of  the  species,  while  others 
are  perhaps  to  be  considered  simply  as  excretory  products.  The 
odorous  volatile  products  given  off  by  flowers  are  supposed. to  be 
useful  to  the  plant  in  attracting  insects  by  which  cross-fertilization 
is  effected.  The  various  poisonous  substances  stored  up  in  leaves 
and  bark  may  serve  to  protect  the  plant  from  enemies,  etc. 

The  minute  plants  with  which  we  are  especially  concerned  also 
produce  a  great  variety  of  substances,  some  of  which  may  be  useful 
to  the  species  in  the  struggle  for  existence.  Thus  the  deadly  pto- 
maines produced  by  some  of  the  pathogenic  bacteria  serve  to  para- 
lyze the  vital  resisting  power  of  living  animals  and  enable  the  para- 
sitic invader  to  thrive  at  the  expense  of  its  host.  In  the  present 
section  we  shall  consider  in  a  general  way  these  various  products  of 
bacterial  growth. 

Pigment  Production. — A  considerable  number  of  species  are 
distinguished  by  the  formation  of  pigment  of  various  colors  and 
shades.  We  have  all  of  the  shades  of  the  spectrum  from  violet  to 
red.  The  color,  as  a  rule,  is  only  produced  in  the  presence  of  oxy- 
gen, and  when  the  pigment-producing  microorganisms  are  massed 
upon  the  surface  of  a  solid  culture  medium  the  pigment  production 
is  often  limited  to  the  superficial  portion  of  the  mass.  In  some 
cases  a  soluble  pigment  is  formed  which  is  absorbed  by  the  transpa- 


134  PRODUCTS    OF    VITAL    ACTIVITY. 

rent  culture  medium,  coloring  especially  the  upper  portion,  in  stab 
cultures  in  nutrient  gelatin  or  agar.  This  is  the  case  with  Bacillus 
pyocyaneus,  which  produces  a  blue  pigment  which  has  been  isolated 
and  carefully  studied  by  Gessard  and  others.  The  pigment,  which 
is  called  pyocyaniii,  is  soluble  in  chloroform  and  crystallizes  from  a 
pure  solution  in  long  blue  needles.  Acids  change  the  blue  color  to 
red,  reducing  substances  to  yellow.  It  resembles  the  ptomaines  in 
its  chemical  reactions,  being  precipitated  by  platinum  chloride  and 
phosphomolybdic  acid. 

In  some  media  the  color  produced  by  the  Bacillus  pyocyaneus 
(bacillus  of  green  pus)  is  a  fluorescent  green.  The  recent  studies  of 
Gessard  show  that  this  is  a  different  pigment.  According  to  this 
author,  cultures  in  a  two-per-cent  solution  of  peptone  give  a  beautiful 
blue  tint,  the  production  of  which  is  hastened  by  adding  to  the  liquid 
five  per  cent  of  glycerin.  In  nutrient  gelatin  and  agar  cultures  a 
fluorescent  green  color  is  developed,  which,  according  to  Gessard, 
is  due  to  the  presence  of  albumin.  Peptone  and  gelatin  are  said  to 
produce  pyocyaniii  without  the  fluorescent-green  pigment,  and  cul- 
tures in  bouillon  to  give  both  this  and  pyocyanin.  In  milk  the 
fluorescent-green  color  is  first  seen,  but  subsequently,  when  the  ca- 
sein has  been  peptonized  by  a  diastase  produced  in  the  culture,  pyo- 
cyanin is  also  formed.  Several  other  microorganisms  are  known 
which  produce  a  fluorescent-green  color,  due  probably  to  the  same 
pigment  as  is  produced  by  the  bacillus  of  green  pus  in  albuminous 
media. 

Babes  claims  to  have  obtained  two  pigments  from  cultures  of  the 
Bacillus  pyocyaneus  in  addition  to  pyocyanin :  one,  soluble  in  alcohol, 
has  by  transmitted  light  a  chlorophyll-green  color,  by  reflected  light 
it  is  blue;  the  other,  insoluble  in  alcohol  and  chloroform,  by  trans- 
mitted light  is  of  a  dark  orange-red,  by  reflected  light  a  greenish- 
blue. 

In  Gessard's  latest  publication  (1891)  he  shows  that  the  produc- 
tion of  pyocyanin  or  of  the  fluorescent-green  pigment  does  not  de- 
pend alone  upon  the  culture  medium,  but  that  there  are  different 
varieties  of  the  Bacillus  pyocyaneus.  He  has  succeeded  in  producing 
four .  distinct  varieties — one  which  produces  both  pyocyanin  and 
fluorescence,  one  which  produces  pyocyanin  alone,  one  which  pro- 
duces the  fluorescent-green  pigment  alone,  and  one  which  produces 
no  pigment.  The  last-mentioned  non-chromogenic  variety  was  pro- 
duced by  subjecting  the  second  variety  to  the  action  of  heat.  A 
temperature  of  57°  maintained  for  five  minutes  destroyed  the  power 
to  produce  pigment  without  destroying  the  vitality  of  the  bacillus, 
which  was  propagated  through  successive  cultures  without  regaining 
tli  is  power. 


STK;  •  s  HACTKKIOLOGY. 


Plal  t-  II 


Pie  3. 


Sarcina  lutea,a£ar  culture 

iius  prodieiosus.a^ar  culture. 
Bacillus  procyanus,  agaj1  culture. 
Bacillus  Havaniensis, potato  culture. 


PRODUCTS   OF   VITAL   ACTIVITY.  135 

The  well-known  Bacillus  prodigiosus  (also  described  as  a  micro- 
coccus)  produces  a  red  pigment  which  is  insoluble  in  water  but  solu- 
ble in  alcohol.  By  the  addition  of  an  acid  the  color  becomes  car- 
mine and  then  violet,  which  is  changed  to  yellow  by  an  alkali.  The 
color  is  said  by  Schottelius  to  be  diffused  in  the  young  cells,  and 
after  the  death  of  the  cells  to  be  present  in  their  vicinity  in  the  form 
of  granules.  The  same  author  has  shown  that  by  subjecting  the 
bacillus  to  special  conditions  a  variety  may  be  obtained  which  no 
longer  produces  pigment. 

The  conditions  which  govern  the  formation  of  pigment  in  the 
chromogenic  bacteria  are  determined  with  comparative  facility  be- 
cause the  results  of  changed  conditions  are  apparent  to  the  eye  ;  in 
the  case  of  products  which  are  not  colored  the  difficulties  attending 
the  study  of  these  conditions  are  much  greater,  but  the  results  are  in 
many  instances  more  important.  The  following  are  among  the  best 
known  pigment-producing  (chromogenic}  bacteria  : 

Staphylqcoccns  pyogenes  aureus,  Staphylococcus  p}Togenes  cit- 
reus,  Sarcina  aurantiaca,  Sarcina  lutea,  Bacillus  cyanogenus,  Bacillus 
janthinus,  Bacillus  fluorescens  liquefaciens,  Bacillus  indicus,  Bacillus 
pyocyaneus,  Bacillus  prodigiosus,  Spirillum  rubrum. 

Liquefaction  of  Gelatin. — Many  species  of  bacteria,  when 
planted  in  a  medium  containing  gelatin,  cause  a  liquefaction  of  the 
gelatin  in  the  immediate  vicinity  of  the  growing  microorganisms, 
while  many  others  multiply  abundantly  in  the  same  medium  with- 
out liquefying  the  gelatin.  This  character,  as  first  shown  by  Koch, 
is  an  important  one  in  the  differential  diagnosis  of  species  which  re- 
semble each  other  in  form  and  in  other  respects.  It  has  no  relation 
to  pathogenic  power,  as  some  liquefying  organisms  are  harmless  sap- 
rophytes and  some  deadly  disease  germs,  while,  on  the  other  hand, 
non-liquefying  bacteria  may  be  very  pathogenic  or  quite  innocent. 

Liquefaction  is  produced  by  a  soluble  peptonizing  ferment  formed 
during  the  growth  of  the  cells.  This  is  shown  by  the  fact  that  if  a 
liquefying  organism  is  cultivated  in  bouillon  and  the  living  cells  re- 
moved by  filtration  or  killed  by  heat,  the  power  of  liquefying  gelatin 
remains  in  the  culture  fluid.  This  was  first  observed  by  Bitter  (1886) 
and  independently  by  the  writer  in  1887.  In  experiments  made  to 
determine  the  thermal  death-point  of  various  bacteria  the  writer 
found  that  when  cultures  of  liquefying  species  were  subjected  to  a 
temperature  which  killed  the  microorganisms,  a  few  drops  of  the 
culture  added  to  nutrient  gelatin  which  had  been  liquefied  by  heat 
prevented  it  from  subsequently  forming  a  solid  jelly  when  cold. 

In  a  study  of  the  ferments  produced  by  bacteria  which  cause 
liquefaction  of  gelatin — "tryptic  enzymes" — made  by  Fermi,  in  the 


136  PRODUCTS   OF   VITAL   ACTIVITY. 

laboratory  of  the  Hygienic  Institute  of  Munich  (1891),  the  following 
results  were  obtained : 

The  enzymes  were  not  obtained  pure,  and  their  isolation  from 
other  proteids  present  in  the  cultures  was  found  to  be  attended  with 
great  difficulties,  but  their  ferment  action  was  studied  and  was  found 
to  be  influenced  by  various  conditions. 

All  were  destroyed  by  a  temperature  of  70°  C. ,  but  the  enzymes 
produced  by  various  liquefying  bacteria  differed  considerably  as  to 
the  temperature  which  they  were  able  to  withstand.  Some  were  de- 
stroyed by  a  temperature  of  50°  to  55°  C. — Bacillus  megatherium, 
Bacillus  ramosus,  Staphylococcus  pyogenes  aureus  ;  some  by  a  tem- 
perature of  55°  to  60°  C. — Bacillus  subtilis,  Bacillus  pyocyaneus,  Ba- 
cillus fluorescens  liquefacieris,  Sarcina  aurantiaca;  some  by  65°  to 
70°  C. — Bacillus  anthracis,  Spirillum  choleras  Asiatics,  Spirillum  of 
Finkler  and  Prioi\  Spirillum  tyrogenum. 

These  enzymes,  like  the  previously  known  pepsin,  trypsin,  and 
invertin,  do  not  dialyze. 

Only  a  few  of  these  bacteria  enzymes  acted  upon  fibrin,  and  no 
action  was  observed  upon  casein  or  upon  egg  albumen. 

Their  liquefying  action  upon  gelatin  was  prevented  by  the  action 
of  sulphuric  acid,  and  to  a  less  degree  by  nitric  acid,  but  was  not  in- 
terfered with  by  acetic  acid. 

The  liquefying  bacteria,  as  a  rule,  only  produce  enzymes  when 
cultivated  in  a  medium  containing  albumen. 

These  enzymes  are  not  produced  by  a  solution  of  the  protoplasm 
of  dead  bacterial  cells,  but  are  a  product  of  the  vital  activity  of  liv- 
ing cells. 

Among  the  numerous  liquefying  bacteria  known  to  bacteriolo- 
gists we  may  mention  the  following  species  as  deserving  the  student's 
special  attention:  Staphylococcus  pyogenes  aureus,  Staphylococcus 
pyogenes  albus,  Sarcina  lutea,  Sarcina  aurantiaca,  Bacillus  anthra- 
cis, Bacillus  pyocyaneus,  Bacillus  subtilis,  Bacillus  indicus,  Bacillus 
prodigiosus,  Spirillum  cholerse  Asiaticse,  Spirillum  of  Finkler  and 
Prior,  Proteus  vulgaris. 

Fermentation. — The  fermentation  produced  by  various  species  of 
bacteria  in  culture  solutions  containing  saccharose,  glucose,  or  lac- 
tose constitutes  a  valuable  character  for  the  differentiation  of  species. 
While  some  bacteria  give  rise  to  fermentation  in  solutions  contain- 
ing either  of  the  carbohydrates  above  mentioned,  others  break  up  lac- 
tose, but  have  no  effect  upon  glucose  or  saccharose,  and  others  again 
are  without  any  ferment  action.  The  gases  evolved  are  chiefly  car- 
bon dioxide  and  hydrogen.  Ferment  action  may  be  tested  by  adding 
one  to  two  per  cent  of  glucose  to  a  solid  culture  medium — preferably 
agar-agar.  This  is  liquefied  by  heat  in  the  test  tube  containing  it  and 


PRODUCTS   OF   VITAL    ACTIVITY.  137 

a  small  quantity  (one  ose)  of  the  microorganism  to  be  tested  is  intro- 
duced. The  culture  medium  is  then  quickly  solidified  by  placing  the 
test  tube  in  iced  water.  It  is  then  placed  in  the  incubator,  and  when 
colonies  form  bubbles  of  gas  will  be  seen  in  their  vicinity,  if  the  bac- 
terium under  observation  is  able  to  cause  fermentation  of  glucose. 
For  accurate  observations  as  to  the  quantity  and  nature  of  the  gases 
produced  the  fermentation  tube  should  be  used,  as  recommended  by 
Theobald  Smith  (see  Fig.  38). 

Production  of  Acids. — Numerous  bacteria  give  an  acid  reaction 
to  the  media  in  which  they  are  cultivated,  and  the  acids  produced 
are  various — lactic,  acetic,  butyric,  propionic,  succinic,  etc. 

The  power  to  produce  an  acid  is  well  shown  by  adding  to  neu- 
tral or  alkaline  culture  media  a  solution  of  litmus.  The  change  in 
color  due  to  the  formation  of  an  acid  may  be  followed  by  the  eye, 
and  comparative  tests  may  be  made  to  aid  in  the  differentiation  of 
similar  bacteria. 

A  considerable  number  of  bacteria  are  able  to  produce  lactic 
acid  from  milk  sugar  and  other  carbohydrates.  One  of  these  is 
considered  the  special  lactic-acid  ferment — Bacillus  acidi  lactici — and 
is  the  usual  cause  of  the  acid  fermentation  of  milk.  Pure  cultures 
of  this  bacillus  introduced  into  sterilized  milk  or  solutions  of  milk 
sugar,  cane  sugar,  dextrin,  or  mannite,  give  rise  to  the  lactic-acid 
fermentation,  in  which  carbonic  acid  is  also  set  free.  The  process 
requires  free  access  of  oxygen,  and  progresses  most  favorably  at  a 
temperature  of  35°  to  40°  C.,  ceasing  at  about  45°.  In  milk,  coagu- 
lation of  the  casein  occurs  within  fifteen  to  twenty-four  hours  after 
adding  a  small  quantity  of  a  pure  culture  of  the  lactic-acid  bacillus. 
This  is  not  due,  however,  to  the  acid  fermentation,  but  to  a  ferment 
resembling  that  of  rennet,  which  is  produced  by  many  different 
bacteria,  some  of  which  do  not  produce  an  acid  reaction  of  the  milk. 
Among  the  bacteria  which  produce  lactic  acid  from  milk  sugar  we 
may  mention  the  staphylococci  of  pus,  Bacillus  lactis  aerogenes,  and 
Bacillus  coli  communis. 

The  formula  showing  the  transformation  of  sugar  into  lactic 
acid  is  usually  stated  as  follows  :  C6H12O6  —  2(HC3H5O3). 

Acetic  acid  is  also  produced  from  dilute  solutions  of  alcohol  by 
the  action  of  a  special  bacterial  ferment,  which  accumulates  upon 
the  surface  of  the  fluid  as  a  mycoderma,  consisting  almost  entirely 
of  the  Bacillus  aceticus  (Mycoderma  aceti).  Free  access  of  oxygen 
is  required,  and  a  temperature  of  about  33°  C.  is  most  favorable  to 
the  process.  According  to  Duclaux,  the  "  Mycoderma  aceti "  oxi- 
dizes the  alcohol,  in  solutions  containing  it,  so  long  as  any  is  present, 
and  when  it  is  exhausted  it  oxidizes  the  acetic  acid  previously 


138 


PRODUCTS   OF   VITAL   ACTIVITY. 


formed  by  oxidation  of  the  alcohol,  producing  from  it  carbon  diox- 
ide and  water. 

The  formation  of  acetic  acid  from  alcohol  is  shown  by  the  follow- 
ing formula  :  Ethyl  alcohol  CH3.CH2.OH  +  O2  =  CH3.COOH  +  HaO. 

Butyric  acid  is  produced  by  a  considerable  number  of  bacteria, 
one  of  which,  named  Bacillus  butyricus,  has  received  the  special  at- 
tention of  Prazmowski.  This  is  strictly  anaerobic.  In  solutions  of 
starch,  dextrin,  sugar,  or  salts  of  lactic  acid,  when  oxygen  is  ex- 
cluded it  produces  butyric  acid  in  considerable  quantity,  and  at  the 
same  time  carbon  dioxide  and  hydrogen  gas  are  set  free.  Duclaux 
gives  the  f ollowing  formula  of  a  solution  containing  lactate  of  lime 
in  which  the  action  of  the  butyric-acid  ferment  may  be  well  studied  : 


Water, 

Lactate  of  lime  (pure), 
Phosphate  of  ammonia, 
Phosphate  of  potash,. 
Sulphate  of  magnesa, 
Sulphate  of  ammonia, 


8  to  10  litres. 

225  grammes. 

0.75 

0.4 

0.4 

0.2 


FIG.  77. 

This  is  introduced  into  a  flask  with  two  necks,  such  as  is  shown 
in  Fig.  77.  Having  filled  the  flask  with  the  culture  liquid,  the  bent 
neck  is  dipped  into  a  porcelain  dish  containing  the  same.  Heat  is 
then  applied  both  to  flask  and  dish,  and  the  liquid  in  each  is  kept  in 
ebullition  for  half  an  hour.  By  this  means  the  air  is  completely 
driven  out  of  the  flask.  This  is  now  allowed  to  cool,  while  the  fluid 
in  the  shallow  dish  is  kept  hot,  so  that  the  liquid  mounting  from  it 
into  the  flask  shall  be  free  from  air.  When  the  flask  is  full  it  is 
transferred  to  an  incubating  oven  heated  to  25°  to  30°  C.,  and  the  bent 
tube  is  immersed  in  a  dish  containing  mercury.  The  little  funnel 


PRODUCTS    OF   VITAL    ACTIVITY.  139 

attached  to  the  upright  tube  is  then  filled  with  carbon  dioxide  and  a 
culture  of  the  butyric-acid  bacillus  is  introduced  into  the  funnel. 
By  turning  the  stopcock  in  the  upright  tube  a  little  of  the  culture  is 
admitted  to  the  flask  without  admitting  any  air.  Fermentation  com- 
mences very  soon,  as  is  seen  by  the  bubbles  of  gas  given  off.  The 
liquid  loses  its  transparency  and  the  lactic  acid  is  gradually  con- 
sumed, butyrate  of  lime  taking  the  place  of  the  lactate. 

Aerobic  bacilli  capable  of  producing  butyric  acid  in  culture  solu- 
tions containing  grape  sugar  or  milk  sugar  have  also  been  described 
by  Liborius  and  by  Hueppe. 

Fitz  has  shown  that  in  culture  solutions  containing  glycerin  the 
Bacillus  pyocyaneus  produces  butyric  acid  in  addition  to  ethyl  alcohol 
and  succinic  acid.  Bacillus  Fitzianus  also  produces  some  butyric  acid 
in  solutions  containing  glycerin,  although  the  principal  product  of  the 
fermentation  caused  by  this  microorganism  is,  according  to  Fitz, 
ethyl  alcohol,  twenty-nine  grammes  of  which  may  be  obtained  from 
one  hundred  grammes  of  glycerin. 

Botkin  (1892)  has  described  a  "Bacillus  butyricus"  (No.  46G) 
which  he  has  not  been  able  to  identify  positively  with  the  butyric- 
acid  ferment  described  by  Prazmowski.  It  is  a  widely  distributed 
anaerobic  bacillus,  which  he  was  able  to  obtain  from  milk  or  water 
containing  it  by  placing  it  in  the  steam  sterilizer  for  half  an  hour. 
The  spores  resisted  this  temperature  and  subsequently  grew  in  anae- 
robic cultures,  in  a  suitable  medium,  while  all  other  bacteria  and 
spores  present  were  destroyed. 

The  writer  has  described  a  bacillus  which  causes  active  acid 
fermentation  in  culture  solutions  containing  glycerin.  The  acid 
formed  is  volatile  and  is  probably  propionic  acid — see  Bacillus  acidi- 
formans. 

The  Caucasian  milk  ferment — Bacillus  Kaukasicus — produces 
a  variety  of  products  in  the  fermented  milk  which  is  a  favorite 
drink  among  the  Caucasians.  The  principal  ones  are  ethyl  alcohol, 
lactic  acid,  and  carbon  dioxide,  but  in  addition  to  these  small  quanti- 
ties of  succinic,  butyric,  and  acetic  acids  are  formed.  The  inhabi- 
tants of  the  Caucasian  mountains  prepare  this  fermented  drink  in  a 
very  simple  manner  from  the  milk  of  cows  or  goats,  to  which  they 
add  the  dried  ferment  collected  from  a  receptacle  in  which  the  fermen- 
tation had  previously  taken  place.  Flugge  gives  the  following  di- 
rections for  the  preparation  of  this  drink  : 

"  Two  methods  may  be  employed.  In  the  first  the  dry  brown  kefir-kor- 
ner  of  commerce  are  allowed  to  lie  in  water  for  five  to  six  hours  until  they 
swell;  they  are  then  carefully  washed  and  placed  in  fresh  milk,  which 
should  be  changed  once  or  twice  a  day  until  the  korner  become  pure  white 
in  color  and  when  placed  in  fresh  milk  quickly  mount  to  the  surface — in 
twenty  to  thirty  minutes.  One  litre  of  milk  is  then  poured  into  a  flask  and  a 
full  tablespoonful  of  the  prepared  korner  added  to  it.  It  is  allowed  to  stand 


140  PRODUCTS   OP   VITAL   ACTIVITY. 

open  for  five  to  eight  hours  ;  the  llask  is  then  closed  and  kept  at  18°  C.  It 
should  be  shaken  every  two  hours.  At  the  end  of  twenty  four  hours  the 
milk  is  poured  through  a  fine  sieve  into  another  flask,  which  must  not  be 
more  than  four-fifths  full.  This  is  corked  and  allowed  to  stand,  being1 
shaken  from  time  to  time.  At  the  end  of  twenty-four  hours  a  drink  is  ob- 
tained which  contains  but  little  COa  or  alcohol.  Usually  it  is  not  drunk 
until  the  second  day,  when,  upon  standing,  two  layers  are  formed,  the 
lowrer  milky,  translucent,  and  the  upper  containing  fine  flakes  of  casein. 
When  shaken  it  has  a  cream- like  consistence.  On  the  third  day  it  again 
becomes  thin  and  very  acid. 

"  The  second  method  is  used  when  one  has  a  good  kefir  of  two  or  three 
days  to  start  with.  Three  or  four  parts  of  fresh  cow's  milk  are  added  to  one 
part  of  this  and  poured  into  flasks  which  are  allowed  to  stand  for  forty- 
eight  hours  with  occasional  shaking  When  the  drink  is  ready  for  use  a 
portion  (one-fifth  to  one-third)  is  left  in  the  flask  as  ferment  fora  fresh 
quantity  of  milk.  The  temperature  should  be  maintained  at  about  18°  C. ; 
but  at  the  commencement  a  higher  temperature  is  desirable.  The  Jcorner 
should  be  carefully  cleaned  from  time  to  time  and  broken  up  to  the  size  of 
peas.  The  cleaned  korner  may  be  dried  upon  blotting  paper  in  the  sun  or 
in  the  vicinity  of  a  stove:  when  dried  in  the  air  they  retain  their  power  to 
germinate  for  a  long  time." 

Fermentation  of  urea.  The  alkaline-  fermentation  of  urine  is 
effected  by  various  microorganisms,  but  chiefly  by  the  Micrococcus 
urese,  the  ferment  action  of  which  has  been  carefully  studied  by  Pas- 
teur, Duclaux,  and  others.  The  change  which  occurs  under  the 
action  of  the  living  ferment  was  determined  by  the  chemist  Dumas 
as  long  ago  as  1830,  but  it  remained  for  Pasteur  to  show  that  this 
change  depends  upon  the  presence  and  vital  activity  of  a  living 
microorganism. 

The  transformation  of  urea  into  carbonate  of  ammonia  is  shown 
by  the  following  formula  :  COH4N2  +  2H2O  =  CO,  +  2NH3  + 
H20  =  (NH4)2C08. 

According  to  Van  Tieghem,  Micrococcus  ureaB  continues  to  grow 
in  a  liquid  containing  as  much  as  thirteen  per  cent  of  carbonate  of 
ammonia.  It  may  be  cultivated  in  an  artificial  solution  of  urea,  with 
the  addition  of  some  phosphates,  as  well  as  in  urine. 

The  Bacillus  urese  of  Miquel  has  also  the  power  of  producing  the 
alkaline  fermentation  of  urine,  but  it  does  not  thrive  in  so  strong  a 
solution  of  carbonate  of  ammonia. 

A  different  micrococcus — Micrococcus  urese  liquef aciens — nas  also 
been  studied  in  Flugge's  laboratory  which  possesses  the  same  power. 
According  to  Musculus,  a  soluble  ferment  may  be  isolated  from  urine 
which  has  undergone  alkaline  fermentation,  which  changes  urea  into 
carbonate  of  ammonia.  He  obtained  it  from  urine  containing  con- 
siderable mucus,  in  a  case  of  catarrh  of  the  bladder.  But  Leube  has 
shown  that  cultures  of  Micrococcus  urese  from  which  the  micrococ- 
cus was  removed  by  filtration  through  clay  do  not  induce  alkaline 
fermentation.  The  soluble  ferment  obtained  by  Musculus  must 
therefore  be  from  some  other  source. 


PRODUCTS    OF   VITAL   ACTIVITY.  141 

Miquel  has  given  special  attention  to  the  study  of  bacteria  which 
produce  alkaline  fermentation  in  urine,  and  in  addition  to  the  spe- 
cies above  mentioned  has  described  the  following  :  Urobacillus  Pas- 
teuri,  Urobacillus  Duclauxi,  Urobacillus  Freudenreichi,  Urobacillus 
Maddoxi,  Urobacillus  Schutzenbergi. 

Viscous  fermentation.  A  special  fermentation  which  occurs 
sometimes  in  wines,  and  in  the  juices  of  bulbous  roots  containing 
glucose,  and  in  milk,  is  produced  by  various  bacteria.  One  of  these 
is  a  micrococcus  which  has  been  described  by  Conn  under  the  name 
of  Micrococcus  lactis  viscosus.  The  fermented  juices  become  very 
viscous,  owing  to  the  formation  of  a  gum-like  product  resembling 
dextrin;  at  the  same  time  mannite  and  CO2  are  produced.  The 
gum-like  substance,  called  viscose  by  Bechamp,  is  soluble  in  cold 
water  and  is  precipitated  by  alcohol.  Guillebeau  (1892)  has  de- 
scribed a  micrococcus  and  a  bacillus  which  produce  viscous  fer- 
mentation ID  milk — Micrococcus  Freudenreichi  and  Bacillus  Hessi. 
A  micrococcus  producing  viscous  fermentation  in  milk  has  also 
been  described  by  Schmidt-Muhlheim,  and  a  bacillus  by  Lomer. 
Bacillus  mesentericus  vulgatus  also  produces  a  similar  change  in 
milk. 

Marsh  gas,  CH4,  is  produced  by  the  fermentation  of  cellulose, 
through  the  action  of  microorganisms  the  exact  characters  of  which 
have  not  yet  been  determined.  According  to  Tappeiner,  there  are 
two  different  fermentations  of  cellulose.  The  first  occurs  in  a  neu- 
tral one-per-cent  flesh  extract  solution  to  which  cotton  or -paper  pulp 
has  been  added.  The  gases  given  off  are  CO2  and  CH4  and  small 
quantities  of  H2S.  The  second  fermentation  occurs  when  an  alkaline 
solution  of  flesh  extract  containing  cellulose  in  suspension  is  used. 
The  gases  formed  are  CO2  and  H.  In  both  cases  small  quantities  of 
aldehyde,  isobutyric  acid,  and  acetic  acid  are  produced. 

Hydrosulpliuric  acid,  H2S.  This  gas  is  produced  during  the 
growth  of  certain  bacteria.  The  conditions  governing  its  develop- 
ment have  been  studied  by  Holschewnikoff,  who  experimented  with 
two  species,  one  isolated  by  himself  and  one  by  Lindenborn.  named 
respectively  Bacterium  sulfureum  and  Proteus  sulfureus.  The  first- 
mentioned  bacterium,  when  inoculated  into  eggs,  produced  within 
three  or  four  days  an  abundant  quantity  of  H,S  ;  the  other  did  not. 
Upon  rawr  albumin  both  species  produced  but  little,  and  on  the  yolk 
of  egg  a  considerable  amount  of  this  gas.  Upon  cooked  egg  the 
action  was  the  reverse.  In  peptone-bouillon  the  evolution  of  H2S 
was  abundant  :  in  the  absence  of  peptone,  ven~  slight. 

Putrefactive  fermentation.  The  putrefactive  decomposition 
of  albuminous  material  of  animal  and  vegetable  origin  is  effected 
by  a  great  variety  of  microorganisms  and  gives  rise  to  the  forma- 


142  PRODUCTS    OF   VITAL   ACTIVITY. 

tion  of  a  great  variety  of  products,  some  of  which  are  volatile  and 
are  characterized  by  their  offensive  odors.  According  to  Fliigge,  the 
first  change  which  occurs  consists  in  the  transformation  of  the  albu- 
mins into  peptone,  and  this  may  be  effected  by  a  large  number  of 
different  bacteria.  Among  the  products  of  putrefactive  fermenta- 
tion known  to  chemists  are  the  following  substances  :  Carbon  diox- 
ide, hydrogen,  nitrogen,  hydrosulphuric  acid  (H,S),  phosphoretted 
hydrogen  (PH3),  methane,  formic  acid,  acetic  acid,  butyric  acid, 
valerianic  acid,  palmitic  acid,  crotonic  acid,  glycolic  acid,  oxalic 
acid,  succinic  acid,  propionic  acid,  lactic  acid,  amidostearic  acid, 
leucin,  ammonia,  ammonium  carbonate,  ammonium  sulphide,  tri- 
methylamine,  propylamine,  indol,  skatol,  tyrosin,neuridin,  cadaverin, 
putrescin,  cholin,  neurin,  peptotoxin,  and  various  other  volatile" 
acids,  ptomaines,  etc. 

The  special  products  of  putrefaction  vary  according  to  the  nature 
of  the  material,  the  conditions  in  which  it  is  placed,  and  the  micro- 
organisms present.  One  or  the  other  of  the  bacteria  concerned  will 
take  the  precedence  when  circumstances  favor  its  growth.  Thus  the 
aerobic  bacteria  cannot  grow  unless  the  putrefying  material  is  freely 
exposed  to  atmospheric  oxygen  ;  the  anaerobic  species  require  its 
exclusion.  Some  saprophytic  bacteria  grow  at  a  comparatively  low 
temperature,  others  take  the  precedence  when  the  temperature  is 
high  ;  some,  no  doubt,  thrive  only  in  presence  of  products  evolved 
by  other  species,  and  are  consequently  associated  with  and  depend- 
ent upon  these  species  ;  some  are  restrained  in  their  growth  sooner 
than  others  by  the  products  evolved  as  a  result  of  their  own  vital 
activity  or  that  of  associated  organisms  ;  some  grow  in  the  presence 
of  acids  and  give  rise  to  an  acid  fermentation  which  wholly  prevents 
the  development  of  other  species. 

At  the  outset  putrefaction  is  often  attended  with  the  presence 
of  several  species  of  micrococci  and  certain  large  bacilli,  which  are 
displaced  later  by  short  motile  bacteria  belonging  to  a  group  which 
includes  several  bacilli  formerly  described  under  the  common  name 
of  Bacterium  termo. 

The  malodorous  volatile  products  of  putrefaction  are  to  a  consid- 
erable extent  produced  by  anaerobic  species.  For  this  reason  these 
odors  are  more  pronounced  when  masses  of  albuminous  material 
undergo  putrefaction  in  situations  where  the  oxygen  of  the  air  has 
not  free  access,  or  where  it  is  displaced  by  carbon  dioxide.  The 
body  of  a  dead  animal,  although  freely  exposed  to  the  air,  furnishes 
in  its  interior  a  suitable  nidus  for  these  anaerobic  gas-forming  spe- 
cies, and  they  may  give  rise  to  products  of  one  kind,  while  aerobic 
species  upon  the  surface  of  the  mass  induce  different  forms  of  putre- 
factive fermentation.  In  the  bodies  of  living  animals  these  anaero- 


PRODUCTS   OF   VITAL   ACTIVITY.  143 

bic  microorganisms  are  constantl}7  present  in  the  intestine,  and  after 
death  they  quickly  invade  the  body  and  multiply  at  its  expense 
under  favorable  conditions  as  to  temperature.  The  surface  decom- 
position due  to  aerobic  bacteria  occurs  later  and  is  not  attended 
with  the  same  putrefactive  odors,  the  products  evolved  being  of  a 
simpler  chemical  composition — CO2,  HN3.  No  doubt  these  aerobic 
bacteria,  by  consuming  the  oxygen  and  forming  an  atmosphere  of 
carbon  dioxide,  help  to  make  the  conditions  favorable  for  the  con- 
tinued development  of  the  anaerobics  in  the  interior  of  the  organic 
mass  ;  at  the  same  time  they  find  a  suitable  pabulum  in  some  of  the 
more  complex  products  of  decomposition  occurring  in  the  absence 
of  oxygen.  The  gases  produced  in  the  interior  of  a  putrefying  mass 
are  mainly  CH4,  H2S,  and  H. 

Many  of  the  bacteria  of  putrefaction  are  facultative  anaerobics— 
that  is  to  say,  they  are  able  to  multiply  either  in  the  presence  of  oxy- 
gen or  in  its  absence.     The  products  evolved  by  these  differ,  no 
doubt,  according  to  whether  they  are  or  are  not  supplied  with  atmos- 
pheric oxygen. 

The  anaerobic  bacteria  concerned  in  putrefaction  have  as  yet 
received  comparatively  little  attention.  Among  the  aerobics  and 
facultative  anaerobics  the  following  are  best  known:  Micrococcus 
fcetidus,  Bacillus  saprogenes  I.,  II.,  and  III.,  Bacillus  coprogenes 
foetidus,  Bacillus  putrificus  coli,  Proteus  vulgaris,  Proteus  Zenkeri, 
Proteus  mirabilis,  Bacillus  pyogenes  fcetidus,  Bacillus  fluorescens 
liquefaciens,  Bacillus  pyocyaneus,  Bacillus  coli  communis,  Bacillus 
janthinus. 

Soluble  Ferments. — Several  species  of  bacteria  produce  soluble 
ferments  capable  of  changing  starch  into  maltose,  dextrin,  etc. 
Hueppe  has  shown  that  the  lactic-acid  bacillus  produces  a  diastase, 
and  Miller  obtained  from  the  human  intestine  a  species  which  dis- 
solves starch.  Marcano,  by  filtering  cultures  of  species  capable  of 
this  ferment  action  through  porcelain,  was  able  to  show  that  the 
effect  is  due  to  a  soluble  ferment,  which  must  have  been  produced 
by  the  vital  activity  of  the  living  microorganisms.  Wortmann  also 
obtained  a  diastase  from  culture  liquids  which  was  precipitated  by 
alcohol  and  again  dissolved  in  water ;  in  slightly  acid  solutions  it 
promptly  converted  starch  into  glucose.  This  is  said  to  be  produced 
in  culture  liquids  only  when  these  do  not  contain  albumin.  In  the 
presence  of  albumin  a  peptoiiizing  ferment  was  formed  ;  in  its  ab- 
sence, a  diastase  by  which  starch  was  dissolved  to  serve  as  pabulum 
for  the  bacteria  present.  These  experiments  were  not  made  with 
pure  cultures,  and  more  exact  researches  in  this  direction  are  de- 
sirable. 


144  PRODUCTS    OF   VITAL   ACTIVITY. 

A  peptonizing  ferment  for  gelatin  is  produced  by  a  considerable 
number  of  bacteria,  as  stated  under  the  heading  "  Liquefaction  of 
Gelatin."  The  jellified  albumin  in  cultures  in  blood  serum  is  also 
liquefied  by  a  peptonizing  ferment  produced  by  certain  species  of  bac- 
teria. 

Some  authors  also  speak  of  a  soluble  ferment  capable  of  inverting 
cane  sugar  or  milk  sugar.  According  to  Hueppe,  such  a  ferment 
is  produced  by  the  Bacillus  acidi  lactici.  A  soluble  ferment  for  cel- 
lulose is  supposed  by  Fliigge  to  be  produced  by  several  species— 
among  others  by  Bacillus  butyricus  and  by  Vibrio  rugula. 

Several  bacilli  produce  a  soluble  ferment  capable  of  coagulating 
the  casein  of  milk. 

Reduction  of  Nitrates,  and  Nitrification. — The  researches  of 
Gayon,  Dupettit,  and  others  show  that  certain  bacteria  are  able  to 
reduce  nitrates  with  liberation  of  ammonia  and  free  nitrogen.  This 
is  effected  in  the  absence  of  oxygen  by  anaerobic  bacteria,  and, 
among  others,  by  Bacillus  butyricus.  Certain  aerobic  bacteria  also 
accomplish  the  same  result.  Thus  Heraaus  obtained  two  species 
from  water  which  reduced  nitrates  in  a  very  decided  manner.  On 
the  other  hand,  a  number  of  species  are  known  to  oxidize  ammonia, 
producing  nitric  acid.  Schlosing  and  Miinz,  as  a  result  of  numerous 
experiments,  arrived  at  the  conclusion  that  in  the  soil  nitrification  is 
effected  by  a  single  species.  But  it  is  doubtful  whether  they  worked 
with  pure  cultures,  and  more  recent  researches  show  that  several, 
and  probably  many,  different  bacteria  possess  this  power.  Accord- 
ing to  Herseus,  the  following  species,  tested  by  him,  oxidize  am- 
monia :  Bacillus  prodigiosus,  the  cheese  spirillum  of  Deneke,  the 
Finkler-Prior  spirillum,  the  typhoid  bacillus,  the  anthrax  bacillus, 
the  staphylococci  of  pus.  The  oxidation  does  riot  always  go  to  the 
point  of  forming  nitrates,  but  nitrites  may  be  formed  in  the  soil 
(Duclaux).  Warrington.  states  that  certain  bacteria  which  formed 
nitrates  in  a  suitable  culture  medium  produced  only  nitrites  when, 
after  an  interval  of  four  or  five  months,  some  of  the  culture  was 
transferred  to  a  solution  containing  muriate  of  ammonia.  The  same 
author  states  that  the  process  of  nitrification  occurs  only  in  the 
dark. 

The  researches  of  Winogradsky,  of  the  Franklands,  and  of  Jor- 
dan show  that  the  failure  of  earlier  investigators  to  obtain  the  nitri- 
fying bacteria  from  the  soil  in  pure  cultures  was  due  to  the  fact  that 
these  bacteria  do  not  grow  in  the  usual  culture  media.  By  the  use 
of  certain  saline  solutions  the  authors  named  have  succeeded  in  iso- 
lating nitrifying  bacteria  in  pure  cultures,  or  nearly  so.  It  is  still 
uncertain  whether  these  investigators  have  obtained  the  same  bac- 
teria, but  the  microorganisms  described  by  them,  and  obtained  from 


PRODUCTS    OF    VITAL   ACTIVITY.  145 

widely  distant  sources,  are  similar  in  their  morphological  and  bio- 
logical characters,  and  at  least  belong  to  the  same  group.  In  a  com- 
munication made  in  1891  Winogradsky  arrives  at  the  conclusion 
that  the  ferments  which  cause  the  oxidation  of  ammonia  and  pro- 
duction of  nitrites  are  not  capable  of  producing  nitrates,  but  that 
other  microorganisms  are  concerned  in  the  oxidation  of  nitrites. 
In  sterilized  soil  to  which  a  pure  culture  of  his  nitromonas  was 
added  nitrites  only  were  produced,  and  the  presence  of  various 
microorganisms  common  in  the  soil  did  not  result  in  the  forma- 
tion of  nitrates  so  long  as  the  specific  ferment  was  absent  to  which 
this  second  oxidation  is  ascribed  (nitrifying  bacillus  of  Winograd- 
sky). 

Phosphorescence. — Several  different  bacteria  have  been  studied 
which,  in  pure  cultures,  give  rise  to  phosphorescence  in  the  medi- 
um in  which  they  are  cultivated.  In  gelatin  cultures  the  light 
is  sufficient  in  some  instances  to  enable  one  to  tell  the  time  by  a 
watch  in  a  perfectly  dark  room,  and  such  cultures  have  even  been 
photographed  by  their  own  light. 

The  phosphorescence  is  influenced  by  changes  in  the  culture 
medium  and  by  conditions  of  temperature,  but  we  have  no  exact 
knowledge  of  the  mode  of  its  production.  The  Bacillus  phosphores- 
cens  from  sea  water  in  the  vicinity  of  the  West  Indies  gives  the 
most  striking  results,  especially  when  planted  upon  the  surface  of 
cooked  fish  and  placed  in  an  incubating  oven  at  30°  C.  Two  other 
species  have  been  studied  by  Fischer — one  obtained  from  the  water 
of  the  harbor  at  Kiel,  and  the  other  a  widely  distributed  species 
called  by  Fischer  Bacterium  phosphorescens.  Katz  (1891)  has  de- 
scribed several  species  obtained  by  him  from  sea  water  and  from 
phosphorescent  fish  in  the  markets  at  Sydney,  New  South  Wales — 
Bacillus  smaragdino-phosphorescens,  Bacillus  argenteo-phosphores- 
cens,  Bacillus  cyaneo-phosphorescens,  Bacillus  argenteo-phosphores- 
cens  liquefaciens. 
10 


V. 

PTOMAINES    AND    TOXALBUMINS. 

VARIOUS  basic  substances  containing  nitrogen,  and  in  chemical 
constitution  resembling  the  vegetable  alkaloids,  have  been  isolated 
by  chemists  from  putrefying  material  and  from  cultures  of  the  bac- 
teria concerned  in  putrefaction,  and  also  from  certain  pathogenic 
species.  Some  of  these  ptomaines  are  non-toxic,  and  others  are 
very  poisonous  in  minute  doses  (toxines).  The  toxic  substances 
sometimes  developed  in  milk,  cheese,  sausage,  etc.,  are  also  of  this 
nature,  and  are  doubtless  produced  by  the  action  of  microorganisms. 
The  pathogenic  power  of  .the  bacteria  which  cause  various  infectious 
diseases  in  man  and  the  lower  animals  has  also  been  shown  to  result 
from  the  production  of  toxic  ptomaines  or  of  toxalbumins.  Selmi  first 
gave  the  name  ptomaines  to  cadaveric  alkaloids  isolated  by  him,  and 
Panum  subsequently  called  attention  to  the  fact  that  poisonous  basic 
substances  of  this  class  are  contained  in  putrefying  material.  Ex- 
tended researches  with  reference  to  the  ptomaines  have  since  been 
made  by  numerous  chemists,  the  most  important  being  those  of  Berg- 
mann,  Schmiedeberg,  Zuelzer  and  Sonnenschein,  Hager,  Otto,  Sel- 
mi, Brieger,  Gautier  and  Etard,  and  Vaughan. 

For  a  full  account  of  the  history  and  chemical  composition  of  the 
ptomaines  the  reader  is  referred  to  the  valuable  work  of  Vaughan 
and  Novy  ("  Ptomaines  and  Leucomaines,"  Philadelphia,  1891).  In 
the  present  volume  we  shall  give  a  brief  account  only  of  some  of  the 
most  important. 

NON-TOXIC   PTOMAINES. 

Neuridin,  C5HJ4N2. — This  is  one  of  the  most  common  of  the  al- 
kaloids of  putrefaction  and  was  isolated  by  Brieger  in  1884.  It  is 
obtained  most  abundantly  from  tissues  containing  gelatin.  Very 
soluble  in  water,  but  insoluble  in  ether  and  absolute  alcohol.  Has  a 
disagreeable  odor. 

Cadaverin,  C5H14N2. — Isomeric  with  neuridin ;  has  a  very  dis- 
agreeable odor  ;  forms  a  thick,  transparent,  syrupy  liquid  ;  is  vola- 
tile, and  can  be  distilled  with  steam  without  undergoing  decomposi- 
tion. When  exposed  to  the  air  the  base  absorbs  carbon  dioxide  and 


PTOMAINES    AND    TOXALBUMINS.  147 

forms  a  crystalline  mass.  Is  produced  in  cultures  of  the  cholera 
spirillum  and  of  the  spirillum  of  Finkler  and  Prior  which  have  been 
kept  for  a  month  or  more  at  37°  C. 

Putrescin,  C4H]2N2. — A  base  resembling  cadaverin  and  com- 
monly associated  with  it.  Obtained  by  Brieger  from  various  sources, 
most  abundantly  from  substances  containing  gelatin  and  in  the 
more  advanced  stages  of  putrefaction.  It  is  obtained  in  the  form  of 
a  hydrate,  which  is  a  transparent  liquid  having  a  boiling  point  of 
about  135°.  With  acids  it  forms  crystalline  salts. 

Saprin,  C5A16N2. — Resembles  cadaverin  and  is  commonly  as- 
sociated with  it  in  putrefying  material.  Isolated  by  Brieger. 

Methylamine,  CH3.NH2. — Obtained  by  Brieger  from  putrefying 
fish  and  from  old  cultures  of  the  cholera  spirillum. 

Dimetliijlamine,  (CHt),.NH. — Obtained  by  Brieger  from  putre- 
fying gelatin  and  by  Bocklisch  from  decomposing  fish. 

Trimethylamine,  (CH3)3N. — Obtained  from  various  sources,  and 
by  Brieger  from  cultures  of  the  cholera  spirillum  and  of  the  strepto- 
coccus of  pus. 

TOXIC   PTOMAINES. 

Neurin,  C5H13NO. — First  obtained  by  Liebreich  in  1865  as  a 
decomposition  product  of  protagon  from  the  brain.  Obtained  by 
Brieger  from  putrefying  muscular  tissue.  When  crystallized  from 
an  aqueous  solution  it  forms  five-  or  six-sided  plates  ;  from  an  alco- 
holic solution  it  crystallizes  in  the  form  of  needles  (Liebreich).  This 
base  is  toxic  in  small  doses.  In  frogs  the  injection  of  a  few  milli- 
grammes produces  paralysis  of  the  extremities.  Respiration  is  first 
arrested  and  the  heart  stops  in  diastole.  Atropine  appears  to  be  a 
physiological  antidote  to  the  toxic  effects  of  neurin.  In  rabbits  it 
produces  profuse  salivation.  The  pupil  is  contracted  by  the  direct 
application  of  a  concentrated  solution. 

Cholin,  C5H15NX)2. — First  obtained  from  hog's  bile  by  Strecker 
in  1862.  Has  been  obtained  by  Brieger  from  various  sources,  in- 
cluding cultures  of  the  cholera  spirillum.  It  is  also  found  widely 
distributed  in  the  vegetable  kingdom.  May  be  prepared  from  the 
yolk  of  eggs  by  the  method  of  Diakonow.  Cholin  is  obtained  in  the 
form  of  a  syrupy,  alkaline  liquid  which  combines  with  acids  to  form 
deliquescent  salts.  At  first  this  base  was  not  supposed  to  have  toxic 
properties,  but  more  recent  researches  have  shown  that  in  compara- 
tively large  doses  it  produces  symptoms  resembling  those  caused  by 
minute  doses  of  neurin. 

Muscarin,  C5H]5NO3. — This  toxic  principle  of  poisonous  mush- 
rooms has  also  been  obtained  by  Brieger  from  putrefying  fish.  It  may 
be  produced  artificially  by  the  oxidation  of  cholin.  In  small  doses 
it  kills  rabbits  and  frogs.  In  the  rabbit  it  produces  lacrymation  and 


148  PTOMAINKS    AND    TOXALBUMIXS. 

salivation,  the  pupil  is  contracted,  and  the  animal  dies  in  convul- 
sions. Frogs  are  completely  paralyzed  by  the  action  of  muscarin 
and  die  with  arrest  of  the  heart's  action  in  diastole. 

Peptotoxin. — The  exact  composition  of  this  ptomaine  has  not 
been  determined.  Brieger  obtained  it  during  the  early  putrefac- 
tion of  proteid  substances  and  also  from  the  artificial  digestion  of 
fibrin.  It  is  very  poisonous  for  frogs,  which  become  paralyzed  and 
die  within  fifteen  or  twenty  minutes  after  the  subcutaneous  injection 
of  a  few  drops  of  a  dilute  solution.  Rabbits  also  are  killed  by  doses 
of  half  a  gramme  to  a  gramme,  the  symptoms  being  paralysis  of  the 
posterior  extremities  and  stupor.  Peptotoxin  is  soluble  in  water, 
but  insoluble  in  ether  or  chloroform.  It  is  not  destroyed  by  boiling. 

Tyrotoxicon. — First  obtained  by  Yaughan  in  poisonous  cheese, 
and  subsequently  by  the  same  chemist  and  others  in  poisonous  milk 
mid  ice  cream.  Chemically  tyrotoxicon  is  very  unstable.  It  is  de- 
composed when  heated  with  water  to  90°  C.  It  is  insoluble  in  ether. 
From  sixteen  kilogrammes  of  poisonous  cheese  Vaughan  obtained 
0. 5  gramme  of  the  poison.  The  symptoms  produced  in  man  by  eat- 
ing cheese  or  milk  containing  tyrotoxicon  are  vertigo,  nausea,  vomit- 
ing, and  severe  rigors,  with  pain  in  the  epigastrium,  cramps  in  the 
legs,  griping  pain  in  the  bowels  attended  with  purging,  numbness 
and  a  pricking  sensation  in  the  limbs,  and  great  prostration. 

Methyl-guanidin,  C2H7N3. — Obtained  by  Brieger  from  putrefy- 
ing horseflesh  which  had  been  kept  at  a  low  temperature  for  several 
months.  This  base  was  previously  known  to  chemists,  having  been 
obtained  by  the  oxidation  of  creatin.  By  Bocklisch  it  has  been  ob- 
tained from  impure  cultures  of  the  Finkler-Prior  spirillum  which 
had  been  kept  for  about  a  month.  It  is  obtained  as  a  colorless  mass 
having  an  alkaline  reaction,  and  which  is  quite  deliquescent.  Brie- 
ger gives  the  following  account  of  the  toxic  action  as .  tested  on 
guinea-pigs  in  a  dose  of  0. 2  gramme  :  The  respiration  increases  in 
rapidity,  the  pupils  dilate  to  the  extreme  limit,  the  animal  has  copi- 
ous discharges  of  urine  and  faeces,  the  extremities  become  paralyzed, 
and  at  the  end  of  about  twenty  minutes  death  occurs  in  convulsions. 

Mytilotoxin. — Obtained  by  Brieger  from  poisonous  mussels. 
The  toxic  action  resembles  that  of  curare. 

Typhotoxin,  C7H17NO2. — Obtained  by  Brieger  from  bouillon 
cultures  of  the  typhoid  bacillus  which  had  been  kept  for  a  week  or 
more  at  a  temperature  of  about  37.5°  C.  In  mice  and  guinea-pigs 
this  base  produces  salivation,  rapid  respiration,  dilatation  of  the 
pupils,  diarrhoea,  and  death  in  from  twenty-four  to  forty-eight  hours. 
It  is  believed  by  Brieger  that  the  specific  action  of  the  typhoid  bacil- 
lus is  due  to  the  production  of  this  ptomaine. 

A  base  which  is  isomeric  with  typhotoxin  has  been  obtained  by 


PTOMAINES   AND    TOXALBUMINS.  14!) 

Brieger  from  putrefying  horseflesh  which  was  kept  at  a  low  tempe- 
rature for  several  months.  Unlike  it,  however,  the  free  base  has 
an  acid  reaction,  while  typhotoxin  is  strongly  alkaline.  It  differs  also 
in  its  physiological  action,  being  more  toxic  and  producing  convul- 
sions ;  the  heart  is  arrested  in  diastole.  Typhotoxin,  on  the  other 
hand,  does  not  induce  convulsions  and  the  heart  is  arrested  in  systole. 

Tetanin,  C13H30N2O4. — Obtained  by  Brieger  from  impure  cul- 
tures of  the  tetanus  bacillus  cultivated  in  bouillon  in  an  atmosphere 
of  hydrogen.  (The  tetanus  bacillus  is  a  strict  anaerobic. )  Obtained 
subsequently  by  the  same  chemist  from  the  amputated  arm  of  a  pa- 
tient with  tetanus.  This  base  has  been  obtained,  by  crystallization 
from  hot  alcohol,  in  clear  yellow  plates  which  are  not  very  soluble  in 
water.  The  hydrochloride  is  a  deliquescent  salt  which  dissolves 
readily  in  alcohol.  When  injected  into  guinea-pigs  or  mice  in  rather 
large  doses,  tetanin  first  causes  the  animal  to  fall  into  a  lethargic 
condition,  followed  by  increased  rapidity  of  respiration  and  tetanic 
convulsions.  In  guinea-pigs  opisthotonos  is  induced,  together  with 
the  characteristic  tetanic  convulsions  as  seen  in  animals  suffering  from 
tetanus.  Three  other  toxic  bases  have  been  obtained  by  Brieger 
from  cultures  of  the  tetanus  bacillus,  which  cause  similar  symptoms. 
One — tetanotoxin — is  given  by  Brieger  the  formula  C.HnN.  A 
second  base,  the  composition  of  which  has  not  been  determined,  is 
called  spasmotoxin. 

Cholera  Ptomaines. — Brieger  has  obtained  from  pure  cultures 
of  the  cholera  spirillum  several  of  the  toxic  ptomaines  heretofore  re- 
ferred to — cadaverin,  putrescin,  cholin,  methyl-guanidin.  In  addi- 
tion to  these  he  found  two  toxic  substances  which  appear  to  be  pe- 
culiar products  of  this  microorganism.  One  induces  cramps  and 
muscular  tremors  in  small  animals,  the  other  diarrhoea  and  symp- 
toms of  collapse. 

Toxalbmnins. — Researches  by  Brieger  and  Frankel  (1890)  show 
that  very  toxic  substances  of  a  different  nature  are  present  in  cultures 
of  some  of  the  pathogenic  bacteria;  these  have  been  designated  by  the 
authors  named  "  toxalbumins." 

Roux  and  Yersin  had  previously  shown  that  filtered  cultures  of  the 
diphtheria  bacillus  contain  a  toxic  substance  which  produces  paralysis 
and  death  in  guinea-pigs  and  rabbits.  This  substance  has  now  been 
obtained  in  a  pure  state  and  its  toxic  action  tested  by  the  authors 
first  named.  It  is  destroyed  by  a  temperature  of  60°  C.,  but  remains 
in  an  active  condition  in  cultures  which  have  been  sterilized  by  seve- 
ral hours'  exposure  to  a  temperature  of  50°,  or  in  those  which  have 
been  passed  through  a  clay  filter.  It  is  not  volatile,  and  differs  essen- 
tially from  the  ptomaines  and  also  from  the  soluble  ferments.  It 
was  obtained  as  a  snow-white,  amorphous  mass  which  was  ex- 


150  PTOMAINES    AND    TOX ALBUMINS. 

tremely  toxic  in  its  action  upon  small  animals.  When  injected  into 
guinea-pigs  in  the  proportion  of  two  and  one-half  milligrammes  to 
one  kilogramme  of  body  weight,  it  caused  death  after  a  considerable 
interval  of  time  (from  a  few  days  to  several  weeks),  during  which 
the  animal  became  emaciated  and  spreading  abscesses  and  necrosis 
of  the  tissues  occurred  at  the  point  of  injection.  This  toxalbumin 
was  obtained  in  a  pure  state  by  repeated  precipitation  from  an  aque- 
ous solution  by  means  of  alcohol.  It  is  produced  most  abundantly 
in  cultures  containing  albumin,  and  old  cultures  are  more  toxic  than 
recent  ones.  Chemical  analysis  gave  the  following  result :  C  45.35, 
H  7.13,  N  16.33,  S  1.39,  O  20.80.  The  authors  remark,  however, 
that  the  chemical  characters  have  not  yet  been  fully  determined. 

The  same  chemists  have  obtained  toxic  substances  of  a  similar 
nature  from  cultures  of  the  bacillus  of  typhoid  fever,  of  the  tetanus 
bacillus,  of  the  Staphylococcus  aureus,  and  of  the  cholera  spirillum. 
Hankin  had  previously  obtained  a  toxic  "albumose"  from  cultures 
of  the  anthrax  bacillus  by  precipitation  with  alcohol,  drying,  solu- 
tion in  water,  and  filtration  through  porcelain ;  and  Christmas  had 
obtained  an  albuminous  substance  from  cultures  of  Staphylococcus 
aureus  which  produced  pus  formation  when  injected  beneath  the 
skin  of  rabbits  or  into  the  anterior  chamber  of  the  eye. 

According  to  Brieger  and  Frankel,  these  toxalbumins  are  divided 
into  two  principal  groups,  one  of  which  is  characterized  by  solubility 
in  water,  as  in  that  produced  by  the  diphtheria  bacillus  ;  and  one  in 
which  the  albumin  is  insoluble  or  but  slightly  soluble,  as  is  the  case 
with  those  obtained  from  cultures  of  the  typhoid  bacillus,  the  cholera 
spirillum,  and  the  Staphylococcus  aureus. 

The  toxalbumin  from  cholera  cultures,  obtained  as  pure  as  pos- 
sible and  suspended  in  water,  when  injected  under  the  skin  of  a 
guinea-pig,  caused  its  death  in  two  or  three  days.  It  was  not,  how- 
ever, toxic  for  rabbits,  even  when  injected  in  considerable  quantity. 

On  the  contrary,  the  toxalbumin  of  the  typhoid  bacillus,  which  is 
dissolved  with  difficulty  in  water,  was  more  poisonous  for  rabbits 
than  for  guinea-pigs.  When  injected  subcutaneously  into  rabbits 
death  usually  occurred  in  eight  to  ten  days.  No  notable  pathologi- 
cal changes  were  observed  at  the  autopsy. 

The  toxalbumin  of  Staphylococcus  aureus  killed  rabbits  and 
guinea-pigs  within  a  few  days,  and  in  some  cases  at  the  end  of 
twenty-four  hours.  The  post-mortem  appearances  were  necrosis  or 
purulent  breaking  down  of  the  tissues  at  the  point  of  injection,  with 
swelling  and  redness  of  the  surrounding  tissues  and  general  inflam- 
matory appearances.  The  toxalbumin  of  anthrax  cultures  resembles 
that  of  the  diphtheria  bacillus  in  being  soluble  in  water.  It  was 
obtained  by  Brieger  from  the  organs  of  animals  recently  dead  from 


PTOMAINES   AND   TOXALBUMINS. 


151 


anthrax.     In  a  dry  condition  it  has  a  grayish-white  color  and  gives 
the  reactions  of  albumins. 

The  toxalbumin  of  the  tetanus  bacillus  is  also  soluble  in  Avater. 
It  is  best  obtained  in  bouillon  cultures  containing  glucose. 

G.  and  F.  Klemperer  (1891)  have  announced  their  success  in 
obtaining  a  toxalbumin  from  cultures  of  Micrococcus  pneumonia? 
croupossB  ('diplococcus  pneumonia?') ;  this  they  propose  to  call  pneu- 
motoxin. 

Some  recent  authors  prefer  the  name  toxins  for  the  poisonous 
products  of  bacterial  growth  designated  by  Brieger  and  others  as 
"toxalbumins."  This  avoids  any  definite  statement  as  to  their 
chemical  composition,  which  appears  to  be  still  in  doubt.  The 
poisonous  precipitates  obtained  from  cultures  of  the  tetanus  or  the 
diphtheria  bacillus  give  the  reactions  of  an  albumin  or  albumose 
(Martin),  but  it  is  possible  that  the  toxic  substance  is  simply  as- 
sociated with  bodies  of  this  class,  and  that  they  have  not  yet  been 
isolated  in  a  pure  state.  These  toxins  in  some  cases  are  intimately 
associated  with  the  bacterial  cell — intracellular  toxins — and  their 
toxic  effects  are  exhibited  when  small  quantities  of  dead  bacteria 
are  introduced  into  a  susceptible  animal.  The  extracellular  toxins 
are  better  known,  and  may  be  obtained  from  filtered  culture  solutions 
by  precipitation  with  strong  alcohol.  In  this  case  they  are  associated 
with  the  proteids  which  may  have  been  present  in  the  culture.  The 
fact  that  a  considerable  interval  elapses — twenty-four  hours  to  several 
da}Ts — after  the  injection  of  these  toxins  into  a  rabbit  or  a  guinea-pig 
before  death  occurs,  has  given  rise  to  the  inference  that  these  sub- 
stances are  of  the  nature  of  enzymes  or  ferments.  This  view  is  also 
supported  by  the  ver}"  minute  quantity  required  to  produce  a  fatal 
result.  According  to  Vaillard  a  dose  of  0.00025  gramme  of  the  tet- 
anus toxin  is  sufficient  to  kill  a  guinea-pig. 

Indol  Production. — Numerous  species  of  bacteria,  as  a  result  of 
their  vital  activity,  give  rise  to  the  production  of  indol.  This  may 
be  detected  by  cultivation  in  "  Dunham's  solution "  of  peptone 
(dried  peptone,  I  part;  sodium  chloride,  0.5  per  cent;  distilled  water, 
100  parts).  Upon  adding  a  drop  of  yellow  nitric  acid  to  ten  cubic 
centimetres  of  a  culture  in  this  medium  the  presence  of  indol  will  be 
revealed  by  the  development  of  a  rosy  red  color.  The  presence  of 
nitrous  acid  in  the  yellow  nitric  acid  is  essential  for  the  reaction, 
which,  however,  may  be  obtained  with  pure  nitric  or  sulphuric  acid 
if  a  small  quantity  of  potassium  nitrate  is  added  to  the  culture — one 
cubic  centimetre  of  a  0.2-per-cent  solution. 

"Koch's  Tuberculin."  -This  is  a  glycerin  extract  of  the  toxic 
substances  present  in  cultures  of  the  tubercle  bacillus.  Crude  tu- 
berculin is  obtained  from  liquid  cultures  made  in  veal  broth  to  which 


152 


PTOMAINES    AND    TOXALBUMINS. 


one  per  cent  of  peptone  and  four  to  five  per  cent  of  glycerin  have 
been  added.  This  culture  liquid  is  placed  in  flasks  and  inoculated 
upon  the  surface  with  small  masses  from  a  pure  culture  of  the  tu- 
bercle bacillus.  A  tolerably  thick  and  dry  white  layer  is  developed, 
which  after  a  time  covers  the  entire  surface.  At  the  end  of  six  to 
eight  weeks  development  ceases  and  the  culture  liquid  is  evaporated 
over  a  water  bath  to  one-tenth  its  volume  ;  this,  after  being  filtered, 
constitutes  the  crude  tuberculin.  By  precipitation  with  sixty-per- 
cent alcohol  Koch  has  obtained  from  this  a  white  precipitate  which 
has  the  active  properties  of  the  glycerin  extract.  This  is  soluble  in 
water  and  in  glycerin,  and  has  the  chemical  reactions  of  an  albumi- 
nous body. 

Zuelzer  has  (1891)  reported  his  success  in  isolating  a  toxic  sub- 
stance from  tubercle  cultures.  The  contents  of  tubes  containing 
pure  cultures  of  the  bacillus  are  first  treated  with  hot  water 
acidulated  with  hydrochloric  acid.  This  solution  is  filtered,  evapo- 
rated, and  then  several  times  precipitated  with  platinum  chloride. 
The  double  salt  formed  is  decomposed  by  hydrosulphuric  acid, 
after  which  the  liquid  is  filtered  and  evaporated  to  dryness.  A 
white,  crystalline  salt  is  thus  obtained  which  is  soluble  in  hot  water. 
This  salt  was  toxic  for  rabbits  and  guinea-pigs  in  doses  of  from  one 
to  three  centigrammes.  Death  usually  occurred  in  from  two  to  four 
days.  In  guinea-pigs  one  centigramme  injected  subcutaneously 
caused,  within  a  few  minutes,  a  greatly  increased  frequency  of  respi- 
ration, an  elevation  of  temperature,  and  protrusion  of  the  eyeballs. 

Malleln. — Kalwing,  Preusse,  and  Pearson  have  obtained  from 
cultures  of  the  glanders  bacillus  a  "lymph"  which  somewhat  re- 
sembles the  crude  tuberculin  of  Koch.  This  was  obtained  by 
Preusse  by  treating  old  potato  cultures  of  the  glanders  bacillus  with 
glycerin  and  water.  The  extract  was  filtered  several  times  and  then 
sterilized  in  a  steam  sterilizer.  This  lymph  injected  into  horses  in- 
fected with  glanders  gives  rise  to  a  very  decided  elevation  of  tempe- 
rature, while  in  horses  free  from  this  disease  no  such  result  follows. 


VI. 
INFLUENCE   OF  PHYSICAL  AGENTS. 

Heat. — We  have  already  seen  (Section  II.,  Part  Second)  that  the 
temperature  favorable  for  the  growth  of  most  bacteria  is  between  20° 
and  40°  C. ;  that  some  species  are  able  to  multiply  at  the  freezing  tem- 
perature, and  others  at  as  high  a  temperature  as  60°  to  70°  C. ;  that, 
as  a  rule,  the  parasitic  species  require  a  temperature  of  35°  to  40°; 
and  that  low  temperatures  do  not  kill  bacteria. 

Frisch  (1877)  exposed  various  cultures  to  a  temperature  of  —87°  C., 
which  he  obtained  by  the  evaporation  of  liquid  CO2,  and  found  that 
micrococci  and  bacilli,  after  exposure  to  such  a  temperature,  multi- 
plied abundantly  when  again  placed  in  favorable  conditions.  Prud- 
den  has  also  made  extended  experiments  upon  the  influence  of 
freezing.  He  found  that  while  certain  species  resisted  the  freezing 
temperature  for  a  long  time,  others  failed  to  grow.  Thus  Bacillus 
prodigiosus  did  not  grow  after  being  frozen  for  fifty-one  days  ;  Pro- 
teus vulgaris  was  killed  in  the  same  time,  and  a  slender,  liquefying 
bacillus  obtained  from  Croton  aqueduct  water  was  killed  in  seven 
days.  Staphylococcus  pyogenes  aureus  withstood  freezing  for  sixty- 
six  days,  a  fluorescent  bacillus  from  Hudson  River  ice  for  seventy- 
seven  days,  and  the  bacillus  of  typhoid  fever  for  one  hundred  and 
three  days.  Cultures  made  at  intervals  showed,  however,  a  dimi- 
nution in  the  number  of  bacteria.  A  similar  diminution  would  per- 
haps have  occurred  in  old  cultures  in  which  the  pabulum  for  growth 
was  exhausted,  independently  of  freezing  ;  for  bacteria,  like  higher 
plants,  die  in  time — which  varies  for  different  species — as  a  result  of 
degenerative  changes  in  the  living  protoplasm  of  the  cells,  and  con- 
tinued vitality  in  a  culture  depends  upon  continued  reproduction. 

Repeated  freezing  and  thawing  was  found  by  Prudden  to  be 
more  fatal  to  the  typhoid  bacillus  than  continuous  freezing.  Cul- 
tures were  sterilized  by  being  thawed  out  at  intervals  of  three  days 
and  again  ref rozen,  after  repeating  the  operation  five  times. 

Cadeac  and  Malet  kept  portions  of  a  tuberculous  lung  in  a  frozen 
condition  for  four  months,  and  found  that  at  the  end  of  this  time 
tuberculosis  was  still  produced  in  guinea-pigs  by  injecting  a  small 
quantity  of  this  material. 


154  INFLUENCE   OF   PHYSICAL   AGENTS. 

In  considering  the  influence  of  high  temperatures  we  must  take 
account  of  the  very  great  difference  in  the  resisting  power  of  the 
vegetative  cells  and  the  reproductive  elements  known  as  spores,  also 
of  the  fact  as  to  whether  dry  or  moist  heat  is  used  and  the  time  of 
exposure. 

Dry  Heat. — When  microorganisms  in  a  desiccated  condition  are 
exposed  to  the  action  of  heated  dry  air,  the  temperature  required  for 
their  destruction  is  much  above  that  required  when  they  are  in  a 
moist  condition  or  when  they  are  exposed  to  the  action  of  hot  water 
or  steam.  This  was  thoroughly  demonstrated  by  the  experiments  of 
Koch  and  Wolff hugel  (1881).  A  large  number  of  pathogenic  and 
non-pathogenic  species  were  tested,  with  the  following  general  result : 
A  temperature  of  78°  to  123°  C.  maintained  for  an  hour  and  a  half 
(over  100°  for  an  hour)  failed  to  kill  various  non-pathogenic  bacteria, 
but  was  fatal  to  the  bacillus  of  mouse  septicaemia  and  that  of  rabbit 
septicaemia.  To  insure  the  destruction  of  all  the  species  tested,  in 
the  absence  of  spores,  a  temperature  of  120°  to  128°  C.,  maintained 
for  an  hour  and  a  half,  was  required. 

The  spores  of  Bacillus  anthracis  and  of  Bacillus  subtilis  resisted 
this  temperature  and  required  to  insure  their  destruction  a  tempera- 
ture of  140°  C.  maintained  for  three  hours.  This  temperature  was 
found  to  injure  most  objects  requiring  disinfection,  such  as  clothing 
and  bedding.  But  the  lower  temperature  which  destroys  micro- 
organisms in  the  absence  of  spores  (120°  C.  =.  248°  F.)  can  be  used 
for  disinfecting  articles  soiled  with  the  discharges  of  patients  with 
cholera,  typhoid  fever,  or  diphtheria,  as  the  specific  germs  of  these 
diseases  do  not  form  spores.  It  is  probable  also  that  it  may  be  safely 
used  to  disinfect  the  clothing  of  small-pox  patients,  for  we  have  ex- 
perimental evidence  that  a  lower  temperature  destroys  the  virulence 
of  vaccine  virus  (90°-95°  C. — Baxter). 

In  practical  disinfection  by  means  of  dry  heat  it  will  be  necessary 
to  remember  that  it  has  but  little  penetrating  power.  In  the  experi- 
ments of  Koch  and  Wolffhiigel  it  was  found  that  registering  ther- 
mometers placed  in  the  interior  of  folded  blankets  and  packages  of 
various  kinds  did  not  show  a  temperature  capable  of  killing  bacteria 
after  three  hours'  exposure  in  a  hot-air  oven  at  133°  C.  and  above. 

Moist  Heat. — The  thermal  death-point  of  bacteria,  in  the  ab- 
sence of  spores,  is  comparatively  low  when  they  are  exposed  to  moist 
heat.  The  results  of  the  writer's  experiments  are  given  below : 

"  In  my  temperature  experiments  I  have  taken  great  pains  to  insure  the 
exposure  of  the  test  organisms  to  a  uniform  temperature,  and  have  adopted 
tt-n  minutes  as  the  standard  time  of  exposure.  The  method  employed 
throughout  has  been  as  follows:  From  glass  tubing  having  a  diameter  of 
about  three-sixteenths  of  an  inch  I  draw  out  in  the  flame  of  a  Bunsen  burner 
a  number  of  capillary  tubes,  with  an  expanded  extremity  which  serves  as 


INFLUENCE    OF    PHYSICAL    AGENTS. 


155 


an  air  chamber.  A  little  material  from  a  pure  culture  of  the  test  organ- 
ism is  drawn  into  each  of  these  capillary  tubes  by  immersing-  the  open 
extremity  in  the  culture,  after  having1  gently  heated  the  expanded  end.  The 
end  of  the  tube  is  then  hermetically  sealed  by  heat  These  tubes  are  im- 
mersed in  a  water  bath  maintained  at  the  desired  temperature  for  the  stan- 
dard time.  The  bath  is  kept  at  a  uniform  temperature  by  personal  supervi- 
sion. At  the  bottom  of  the  vessel  is  a  thick  glass  plate  which  prevents  the 
thermometer  bulb  and  capillary  tubes,  which  rest  upon  it,  from  being  ex- 
posed to  heat  transmitted  directly  from  the  bottom  of  the  vessel  To  further 
guard  against  this  I  am  in  the  habit  of  applying  the  flame  to  the  sides  of  the 
vessel,  and  a  uniform  temperature  throughout  the  bath  is  maintained  by 
frequent  stirring1  with  a  glass  rod.  It  is  impossible  to  avoid  slight  variations, 
but  by  keeping  my  eye  upon  the  thermometer  throughout  the  experiment 
I  have  kept  these  within  very  narrow  limits.  .  .  .  No  attempt  has  been  made 
to  fix  the  thermal  death-point  within  narrower  limits  than  2°  C.,  and  in  the 
table  the  lowest  temperature  is  given  which  has  been  found,  in  the  experi- 
ments made,  to  destroy  all  of  the  microorganisms  in  the  material  subjected 
to  the  test.  No  doubt  more  extended  experiments  would  result,  in  some  in- 
stances, in  a  reduction  of  the  temperature  given  as  the  thermal  death-point 
for  a  degree  or  more.  But  the  results  as  stated  are  sufficiently  accurate  for 
all  practical  purposes."  1 

The  results  obtained  in  these  experiments,  for  non-sporebearing 
bacteria,  are  given  in  the  following  table.  The  time  of  exposure 
was  ten  minutes,  except  for  the  cholera  spirillum  and  the  cheese  spi- 
rillum of  Deneke. 

THERMAL    DEATH-POINT   OF   BACTERIA. 


Spirillum  cholene  Asiatic* 52° 

Spirillum  tyrogenum  (cheese  spirillum) 52 

Spirillum  Flakier- Prior 50 

Bacillus  typhi  abdominalis 56 

Bacillus  of  schweine-rothlauf  (rouget) 58 

Bacillus  murisepticus 58 

Bacillus  Neapolitanus  (Emmerich's  bacillus) 62 

Bacillus  cavicida 62 

Bacillus  pneumoniae  (Friedlander's) 56 

Bacillus  crassus  sputigenus 54 

Bacillus  pyocyaueus    56 

Bacillus  iudicus  58 

Bacillus  prodigiosus 58 

Bacillus  cyanogenus 54 

Bacillus  fluorescens .  .    . .  54 

Bacillus  acidi  lactici 56 

Staphylococcus  pyogenes  aureus . .  .  58 

Staphylococcus  pyogenes  citreus 62 

Staphylococcus  pyogenes  albus 62 

Streptococcus  pyogenes 54 

Micrococcus  tetragenus  58 

Micrococcus  Pasteuri 52 

Sarcina  lutea 64 

Sarcina  aurantiaca  62 


Centigrade. 


Fahrenheit. 


125.6C 

125.6 

122. 

138.8 

136.4 

136.4 

143.6 

143.6 

132.8 

129.2 

132.8 

136.4 

136.4 

129.2 

129.2 

132.8 

136.4 

143.6 

143.6 

1292 

136.4 

125.6 

147.2 

143.6 


(4m.) 
(4m.) 


The  following  determinations  of  the  thermal  death-point  of  path- 

1  Quoted  from  the  Report  of  the  Committee  on  Disinfectants  of  the  American  Pub- 
lic Health  Association,  pages  136  and  152. 


156  INFLUENCE    OF   PHYSICAL    AGENTS. 

ogenic  organisms  have  been  made  by  the  authors  named  :  Bacillus 
anthracis  (Chauveau),  54°  C.  ;  Bacillus  mallei — the  bacillus  of  glan- 
ders— (Loffler),  55°  C.,  Bacillus  gallinarum — micrococcus  of  fowl 
cholera— (Salmon),  56°  C.  ;  Bacillus  of  diphtheria  (Loffler),  60°  C. 

In  the  writer's  experiments  the  micrococcus  of  gonorrhoea  was 
apparently  killed  by  exposure  for  ten  minutes  to  a  temperature  of 
60°  C. 

"  Some  gonorrhceal  pus  from  a  recent  case  which  had  not  undergone 
treatment  was  collected  for  me  by  my  friend  Dr.  Rohe  in  the  capillary 
glass  tubes  heretofore  described.  A  microscopical  examination  of  stained 
cover-glass  preparations  showed  that  this  pus  contained  numerous  '  gono- 
cocci '  in  the  interior  of  the  cells.  Two  of  the  capillary  tubes  were  placed 
in  a  water  bath  maintained  at  60°  C.  for  ten  minutes.  The  pus  was  then 
forced  out  upon  two  pledgets  of  cotton  wet  with  distilled  water.  Two 
healthy  men  had  consented  to  submit  to  the  experiment,  and  one  of  these 
bits  of  cotton  was  introduced  into  the  urethra  of  each  and  left  in  situ  for 
half  an  hour.  As  anticipated,  the  result  WHS  entirely  negative.  For  obvi- 
ous reasons  no  control  experiment  was  made  to  fix  the  thermal  death-point 
within  narrower  limits. 

"  In  connection  with  these  experiments  upon  the  thermal  death -point  of 
known  pathogenic  organisms,  it  is  of  interest  to  inquire  whether  the  viru- 
lence of  infectious  material,  in  which  it  has  not  been  demonstrated  that  this 
virulence  is  due  to  a  microorganism,  is  destroyed  by  a  correspondingly  low 
temperature.  Evidently,  if  this  proves  to  be  the  case,  it  will  be  a  strong 
argument  in  favor  of  the  view  that  we  have  to  deal  with  a  microorganism 
in  these  diseases  also.  We  have  experimental  proof  that  a  large  number  of 
pathogenic  organisms  are  killed  by  exposure  for  ten  minutes  to  a  tempera- 
ture of  55°  to  tfO°  C.  But,  s  )  far  as  I  am  aware,  this  low  temperature  would 
not  be  likely  to  destroy  any  of  the  poisonous  chemical  products  which  might 
be  supposed  to  be  the  cause  of  infective  virulence,  leaving  aside  the  fact  that 
such  chemical  products  have  no  power  of  self-multiplication,  and,  there- 
fore, could  not  be  the  independent  cause  of  an  infectious  disease.1 

"  Vaccine  Virus. — Carstens  and  Coert  have  experimented  upon  the  tem- 
perature required  to  destroy  the  potency  of  vaccine  virus.  In  a  paper  read 
at  the  International  Medical  Congress  in  1879  they  report,  as  a  result  of 
their  experiments,  that  the  maximum  degree  of  heat  to  which  fresh  vaccine 
virus  can  be  exposed  without  losing  its  virulence  probably  varies  between 
52°  and  54°  C.  Fresh  animal  vaccine  heated  to  52°  C.  for  thirty  minutes 
does  not  lose  its  virulence.  Fresh  animal  vaccine  heated  to  54. 5 3  for  thirty 
minutes  loses  its  virulence. 

"Rinderpest. — According  to  Semmer  and  Raupach,  exposure  for  ten 
.minutes  to  a  temperature  of  55°  C.  destroys  the  virulence  of  the  infectious 
material  in  this  disease. 

''  Sheep-pox. — The  authors  last  mentioned  have  also  found  that  the  same 
temperature — 55°  C.  for  ten  minutes — destroys  the  virulence  of  the  blood  of 
an  animal  dead  from  sheep-pox. 

"  Hydrophobia. — Desiring  to  fix  the  thermal  death -point  of  the  virus  of 
hydrophobia,  I  obtained,  through  the  kindness  of  Dr.  H.  C.  Ernst,  a  rabbit 
which  had  been  inoculated,  by  the  method  of  trephining,  with  material 
which  came  originally  from  Pasteur's  laboratory.  The  rabbit  sent  me 
showed  the  first  symptom  of  paralytic  rabies  on  the  eighth  day  after  inocu- 
lation. It  died  on  the  eleventh  day  (March  2d,  1887),  and  I  at  once  pro- 
ceeded to  make  the  following  experiment  : 

"  A  portion  of  the  medulla  was  removed  and  thoroughly  mixed  with 

1  Since  this  was  written  Brieger  has  isolated  a  toxalbumin  from  cultures  of  the 
diphtheria  bacillus  which  is  destroyed  by  u  temperature  of  60°  C.,  but  resists  50°. 


INFLUENCE    OF   PHYSICAL   AGENTS.  157 

sterilized  water.  The  milky  emulsion  was  introduced  into  four  capillary 
tubes,  such  as  had  been  used  in  my  experiments  heretofore  recorded.  Two 
of  these  tubes  were  then  placed  for  ten  minutes  in  a  water  bath,  the  tem- 
perature of  which  was  maintained  at  60°  C.  Four  rabbits  were  now  inocu- 
lated by  trephining1,  two  with  the  material  exposed  to  60°  C.  for  ten  min- 
utes, and  two  with  the  same  material  from  the  capillary  tube  not  so  exposed. 
The  result  was  as  definite  and  satisfactory  as  possible.  The  two  control 
rabbits  were  taken  sick,  one  on  March  10th  and  one  on  the  llth  ;  both  died 
with  the  characteristic  symptoms  of  paralytic  rabies  on  the  third  day.  The 
two  rabbits  inoculated  with  material  exposed  to  00°  C.  remained  in  perfect 
health.  On  the  26th  of  March  one  of  these  rabbits  was  again  inoculated, 
by  trephining,  with  material  from  the  medulla  of  a  rabbit  just  dead  from 
hydrophobia.  This  rabbit  died  from  paralytic  rabies  oil  the  8th  of  April. 
Its  companion  remains  in  perfect  health. 

"A  second  experiment  was  made  in  the  same  way  on  the  14th  of  March. 
Two  rabbits  were  inoculated  with  material  exposed  for  ten  minutes  to  a 
temperature  of  50°  C. ;  two  with  material  exposed  to  55°  C. ;  and  two  con- 
trol rabbits  with  material  not  so  exposed.  One  of  the  rabbits  inoculated 
with  material  exposed  to  50°  C.,  and  one  of  the  control  rabbits,  died  on  the 
25th;  the  other  rabbit  inoculated  with  the  material  exposed  to  50°,  the  other 
control,  and  one  inoculated  with  material  exposed  to  55°,  on  the  26th.  The 
second  rabbit  inoculated  with  material  exposed  to  55°  died  five  days  later 
with  the  characteristic  symptoms  of  the  disease-  These  experiments  show, 
then,  that  the  virus  of  hydrophobia  is  destroyed  by  a  temperature  of  603  C., 
and  that  55°  C.  fails  to  destroy  it,  the  time  of  exposure  being  ten  minutes."1 

The  experimental  data  given  show  that  the  pathogenic  bacteria 
tested  and  different  kinds  of  virus  are  all  killed  by  a  temperature  of 
60°  C.  or  below ;  some,  like  the  cholera  spirillum  and  Micrococcus 
pneumonise  crouposse,  failing  to  grow  after  exposure  to  as  low  a  tem- 
perature as  52°  C.  for  four  minutes.  By  extending  the  time  a  still 
lower  temperature  will  effect  the  same  result.  Thus,  according  to 
Chauveau,  the  anthrax  bacillus  is  killed  by  twenty  minutes'  exposure 
to  a  temperature  of  50°  C. ;  and  Brieger  sterilizes  cultures  of  the 
diphtheria  bacillus,  to  obtain  the  soluble  toxalbumin  produced  in 
them,  by  exposure  for  several  hours  to  50°  C.  A  temperature  of  60° 
has  been  found  to  decompose  the  toxalbumin.  The  non-pathogenic 
bacteria  tested  have,  as  a  rule,  a  higher  thermal  death-point — 58°  C. 
for  Bacillus  prodigiosus,  64°  C.  for  Sarcina  lutea,  etc. 

It  is  a  remarkable  fact  that  certain  bacteria  not  only  are  not  de- 
stroyed at  higher  temperatures  than  this,  but  are  able  to  multiply  at 
a  temperature  of  65°  to  70°  C.  Thus  Miquel,  in  1881,  found  in  the 
waters  of  the  Seine  a  motionless  bacillus  which  grew  luxuriantly  in 
bouillon  at  a  temperature  of  69°  to  70°  C.  Van  Tieghem  has  also 
cultivated  several  different  species  at  about  the  same  temperature, 
and  more  recently  Globig  has  obtained  from  the  soil  several  species 
which  grow  at  temperatures  ranging  from  50°  to  70°  C. 

The  resisting  power  of  spores  to  heat  also  varies  in  different  spe- 
cies ;  but  the  spores  of  known  pathogenic  bacteria  are  quickly  de- 
stroyed by  a  temperature  of  100°  C.  (212°  F.).  In  the  writer's  experi- 

1  Report  of  the  Committee  on  Disinfectants  (op.  cit.),  p.  147. 


158  INFLUENCE    OF   PHYSICAL    AGENTS. 

ments  the  spores  of  Bacillus  anthracis  and  of  Bacillus  alvei  failed  to 
grow  after  exposure  to  a  temperature  of  100°  C.  for  four  minutes, 
and  only  a  few  colonies  developed  after  two  minutes'  exposure  to  this 
temperature.  The  thermal  death-point  of  spores  of  the  "  wurtzel  ba- 
cillus "  and  of  Bacillus  butyricus  (of  Hueppe)  was  the  same — 100°  C. 
for  four  minutes. 

Schill  and  Fischer,  in  1884,  made  a  number  of  experiments  to  de- 
termine the  thermal  death-point  of  Bacillus  tuberculosis.  They 
found  that  five  minutes'  exposure  to  a  temperature  of  100°  C.  in 
steam  destroyed  the  vitality  of  the  bacillus  in  sputum  in  five  min- 
utes. When  the  time  was  reduced  to  two  minutes  a  negative  result 
from  inoculation  was  obtained  in  two  guinea-pigs,  but  one  inoculated 
at  the  same  time  became  tuberculous.  My  own  experiments  and 
those  of  Yersin,  made  since,  lead  me  to  think  that  there  may  have 
been  some  cause  of  error  in  this  experiment  of  Schill  and  Fischer, 
and  that  the  thermal  death-point  of  the  spores  of  Bacillus  tuber- 
culosis is  considerably  below  the  boiling  point  of  water.  I  inoculated 
guinea-pigs  with  tuberculous  sputum  subjected  for  ten  minutes  to 
the  following  temperatures  :  50°,  60°,  70°,  80°,  90°  C.  The  animal 
inoculated  with  material  exposed  to  50°  died  from  tuberculosis  at  the 
end  of  seven  weeks.  None  of  the  others  developed  tuberculosis. 

Yersin  exposed  an  old  culture  in  glycerin  bouillon,  in  which  many 
of  the  bacilli  contained  spores — "tres  nettes"  — to  the  following  tem- 
peratures :  55°,  60°,  65°,  70°,  75°,  80°,  85°,  90°,  100°  C.  ' '  At  the  end  of 
ten  days  the  bacilli  heated  to  55°  gave  a  culture  in  glycerin  bouillon  ; 
those  exposed  to  60°  grew  after  twenty-two  days ;  none  of  the 
bacilli  heated  above  70°  gave  any  development.  This  experiment, 
repeated  a  great  number  of  times,  has  always  given  us  the  same  re- 
sult." Voelsh,  who  has  studied  the  same  question,  reports  as  the 
result  of  his  experiments  that  the  tubercle  bacillus  in  sputum  was 
not  destroyed  by  heating  to  100°  C.  Further  experiments  will  be  re- 
quired to  reconcile  these  contradictory  results. 

While  the  spores  of  the  pathogenic  bacteria  mentioned  are  de- 
stroyed by  the  boiling  point  of  water  within  a  few  minutes,  certain 
non-pathogenic  species  resist  this  temperature  for  hours.  Thus 
Globig  obtained  a  bacillus  from  the  soil  the  spores  of  which  required 
five  and  one-half  to  six  hours'  exposure  to  streaming  steam  for  their 
destruction.  These  spores  survived  exposure  for  three-quarters  of  an 
hour  in  steam  under  pressure  at  from  109°  to  113°  C.  They  were  de- 
stroyed, however,  by  exposure  for  twenty-five  minutes  in  steam  at 
113°  to  116°,  and  in  two  minutes  at  127°. 

In  the  practical  application  of  steam  for  disinfecting  purposes  it 
must  be  remembered  that,  while  steam  under  pressure  is  more  effec- 
tive than  streaming  steam,  it  is  scarcely  necessary  to  give  it  the  pre- 


INFLUENCE   OF   PHYSICAL   AGENTS.  159 

ference,  in  view  of  the  fact  that  all  known  pathogenic  bacteria  and 
their  spores  are  quickly  destroyed  by  the  temperature  of  boiling 
water  ;  and  also  that  superheated  steam  is  less  effective  than  moist 
steam.  When  confined  steam  in  pipes  is  '*  superheated  "  it  has  about 
the  same  germicidal  power  as  hot  dry  air  at  the  same  temperature. 
This  is  shown  by  the  experiments  of  Esmarch,  who  found  that  an- 
thrax spores  were  killed  in  streaming  steam  in  four  minutes,  but 
were  not  killed  in  the  same  time  by  superheated  steam  at  a  tempera- 
ture of  141°  C. 

Desiccation. — Cultures  of  bacteria  kept  in  a  moist  condition  re- 
tain their  vitality  for  a  considerable  time,  which  varies  greatly  with 
different  species.  The  writer  has  found  that  a  culture  of  the  typhoid 
bacillus  preserved  in  a  hermetically  sealed  glass  tube  retained  its 
vitality  for  eighteen  months,  as  did  also  Bacillus  prodigiosus,  Bacil- 
lus cavicida,  and  some  others.  According  to  Kitasato,  the  cholera 
spirillum  may  be  preserved  in  a  moist  state  for  seven  months  ;  other 
bacteria  die  out  in  a  month  or  two,  but,  as  a  rule,  vitality  is  preserved 
for  several  months  at  least. 

Spores  in  a  desiccated  condition  preserve  their  vitality  for  a 
great  length  of  time.  But  desiccation  is  quickly  fatal  to  some  of  the 
pathogenic  bacteria,  and  especially  so  to  the  cholera  spirillum.  Koch, 
in  his  earlier  experiments,  found  that  his  "comma  bacillus"  did  not 
grow  after  being  dried  upon  a  cover  glass  for  three  hours.  Kitasato, 
in  experiments  made  since,  found  that  a  bouillon  culture  dried  upon 
a  thin  glass  cover  was  incapable  of  development  after  three  hours' 
time,  but  that  cultures  in  nutrient  agar  or  gelatin  survived  for  two 
days,  probably  on  account  of  the  thicker  layer  formed  and  the  longer 
time  required  for  complete  desiccation.  Pfuhl  has  found  that  the 
typhoid  bacillus  dried  upon  a  cover  glass  retains  its  vitality  for 
eight  to  ten  weeks,  and  Loffler  states  that  the  diphtheria  bacillus  re- 
sists desiccation  for  four  or  five  months.  Cadeac  and  Malet  pro- 
duced tuberculosis  in  guinea-pigs  by  injecting  material  from  the 
lung  of  a  tuberculous  cow  which  had  been  kept  in  the  form  of  a  dried 
powder  for  nearly  five  months  ;  at  a  later  date  the  virulence  was 
lost. 

Light. — Downes  and  Blunt,  in  a  communication  made  to  the 
Royal  Society  of  London  in  1877,  first  called  attention  to  the  fact  that 
light  has  an  injurious  effect  upon  bacteria,  and  that  cultures  may  be 
sterilized  by  exposure  to  direct  sunlight. 

Tyndall,  in  experiments  made  in  the  clear  sunlight  of  the  Alps, 
verified  the  fact  that  the  development  of  bacteria  was  restrained  in 
cultures  during  their  exposure,  but  failed  to  obtain  evidence  that 
vitality  was  destroyed. 

Jn  1885  Duclaux  took  up  the  subject  with  pure  cultures  of  various 


1GO  INFLUENCE   OF   PHYSICAL   AGENTS. 

bacteria,  and  showed  that  by  prolonged  exposure  to  direct  sunlight  the 
spores  of  various  bacilli  lose  their  capacity  to  germinate.  About  the 
same  time  Arloing  published  his  researches  upon  the  influence  of 
light  upon  the  development  of  anthrax  spores.  He  found  that  the 
anthrax  bacillus  was  not  restrained  in  its  growth  by  diffused  lamp- 
light, but  its  growth  was  retarded  by  an  intense  gaslight.  Spore 
formation  was  more  abundant  in  darkness  than  in  red  light,  and  more 
abundant  in  red  than  in  white  light.  When  a  screen  was  interposed 
between  the  culture  and  the  source  of  light,  consisting  of  an  aqueous 
solution  of  hsematoglobin,  the  growth  of  the  bacilli  and  of  spores  was 
much  more  luxuriant  than  in  white  light.  In  yellow  light  it  was  less 
abundant  than  in  red.  The  blue  and  violet  rays  were  still  less  favor- 
able for  the  growth  of  the  bacillus  and  the  development  of  spores. 
The  pathogenic  power  of  cultures  was  not  especially  influenced  by 
exposure  to  white  gaslight.  In  subsequent  experiments  with  sun- 
light Arloing  found  that  two  hours  of  exposure  to  the  July  sun  suf- 
ficed to  destroy  the  vitality  of  anthrax  spores,  but  that  a  considerably 
longer  exposure  (twenty-six  to  thirty  hours)  was  necessary  when  the 
spores  had  bean  allowed  to  germinate  in  a  suitable  culture  medium. 
Cultures  which  were  not  exposed  long  enough  to  destroy  the  vitality 
of  the  bacilli  were  retarded  in  their  growth,  and  subsequent  exposure 
for  a  shorter  time  (nine  to  ten  hours)  completely  sterilized  them. 
Cultures  which  were  weakened  in  their  reproductive  energy  by  ex- 
posure to  sunlight  were  also  ' '  attenuated  "  as  to  their  pathogenic 
power  and  could  be  used  as  a  vaccine  in  protective  inoculations.  Ac- 
cording to  Arloing,  the  effect  produced  results  from  the  action  of  the 
full  sunlight  and  cannot  be  obtained  by  the  use  of  monochromatic 
light. 

The  experiments  of  Strauss  seemed  to  give  support  to  the  view 
advanced  by  Nocard  that  in  Arloing's  experiments  spores  did  not 
really  exhibit  a  less  degree  of  resisting  power  than  the  vegetating 
bacilli,  but  that  in  fact  they  commenced  to  vegetate  before  they  were 
killed.  Strauss  placed  anthrax  spores  in  sterilized  distilled  water  and 
in  bouillon,  and  found  that,  under  the  same  conditions  of  exposure, 
the  bouillon  cultures  were  sterilized  in  direct  sunlight  in  nine 
hours,  while  the  spores  suspended  in  distilled  water  grew  when  trans- 
ferred to  a  suitable  medium.  This  was  accounted  for  on  the  suppo- 
sition that  the  bouillon  furnishes  the  necessary  pabulum  for  the  de- 
velopment of  the  spores  and  that  distilled  water  does  not. 

Arloing  combats  this  view  and  has  published  additional  experi- 
ments which  seem  to  disprove  it.  He  placed  small  flasks  containing 
anthrax  spores  in  bouillon  in  the  direct  rays  of  the  sun  in  February. 
Some  of  the  flasks  were  placed  upon  a  block  of  ice  which  reduced  the 
temperature  to  4°  C. ;  the  others  were  not  so  placed,  and  the  tempe- 


INFLUENCE    OF    PHYSICAL    A(,KNTs.  161 

rature.  in  the  open  air  where  all  were  exposed,  was  11°  C.  All  of  the 
spores  failed  to  grow  after  an  exposure  of  four  hours.  When  exposed 
in  water  the  time  of  exposure  was  longer. 

Roux  has  shown  that  the  light  also  has  an  effect  upon  the  culture 
medium,  and  that  sterilized  bouillon  which  has  been  exposed  to  direct 
sunlight  for  some  hours  restrains  the  development  of  anthrax  spores 
subsequently  introduced  into  it,  but  not  of  the  growing  bacilli.  His 
experiments  show  that  access  of  oxygen  is  a  necessary  factor  in  the 
sterilization  of  cultures  by  sunlight. 

In  the  experiments  of  Moment  (ISO'-?)  dry  anthrax  spores  were 
found  to  resist  the  action  of  light  for  a  long  time,  but  moist  spores, 
freely  exposed  to  the  air,  failed  to  grow  after  forty-four  hours'  ex- 
posure to  sunlight.  In  the  absence  of  spores,  anthrax  bacilli  in  a 
moist  condition,  when  freely  exposed  to  the  air,  failed  to  grow  after 
exposure  to  sunlight  for  half  an  hour  to  two  hours  ;  but  in  the  ab- 
sence of  air  the  same  bacilli  were  not  destroyed  at  the  end  of  fifty 
hours'  exposure. 

Geisler  (1892),  in  experiments  made  upon  the  typhoid  bacillus, 
found  that  all  portions  of  the  solar  spectrum  except  the  red  rays  ex- 
ercised a  restraining  influence  upon  the  development  of  this  bacillus. 
The  electric  light  gave  a  similar  result.  The  most  decided  effect  was 
produced  by  rays  from  the  violet  end  of  the  spectrum.  The  restrain- 
ing influence  appears,  from  the  researches  of  Geisler,  not  to  be  due 
solely  to  the  direct  action  of  light  upon  the  development  of  the 
bacilli,  but  also  to  changes  induced  in  the  gelatin  culture  medium 
employed  in  his  experiments. 

In  his  address  before  the  International  Medical  Congress  of  Berlin, 
1800,  Koch  states  that  the  tubercle  bacillus  is  killed  by  the  action  of 
direct  sunlight  in  a  time  varying  from  a  few  minutes  to  several  hours, 
depending  upon  the  thickness  of  the  layer  exposed.  Diffused  day- 
light also  has  the  same  effect,  although  a  considerably  longer  time  of 
exposure  is  required — when  placed  close  to  a  window,  from  five  to 
seven  days. 

Dieudonne  (1804),  in  experiments  upon  Bacillus  prod igiosus  and 
Bacillus  fluorescens  putidus,  found  that  direct  sunlight  in  March, 
July,  and  August  killed  these  bacilli  in  one  and  one-half  hours,  in 
November  in  two  and  one-half  hours.  Diffuse  daylight  in  March 
and  July  restrained  development  after  three  and  one-half  hours'  ex- 
posure (in  November  four  and  one-half  hours),  and  completely  de- 
stroyed vitality  in  from  five  to  six  hours. 

Ward's  experiments  (180-2-1804)  show  that  the  blue  and  violet 
rays  have  decided  germicidal  power,  while  the  rays  at  the  red  end  of 
the  spectrum  are  comparatively  inert.     This  corresponds  with  results 
previously  reported  by  Arloing. 
11 


162  INFLUENCE   OF   PHYSICAL   AGENTS. 

In  the  writer's  experiments  on  the  cholera  spirillum  (1802)  test 
tubes,  containing  sterile  bouillon  inoculated  with  one  or  two  ose  of  a 
pure  culture,  were  sterilized  by  two  hours'  exposure  to  direct  sunlight 
(in  December). 

Dieudonne  (1894)  found  that  the  electric  arc  light  destroyed  his 
test  organisms  (Bacillus  prod igiosu sand  Bacillus fluorescens  putidus) 
in  eight  hours.  The  same  result  was  accomplished  by  the  incandes- 
cent light  in  eleven  hours. 

In  view  of  these  facts  we  may  conclude,  with  Duclaux,  that  sun- 
light is  one  of  the  most  potent  and  one  of  the  cheapest  agents  for  the 
destruction  of  pathogenic  bacteria,  and  that  its  use  for  this  purpose  is 
to  be  remembered  in  making  practical  hygienic  recommendations. 
The  popular  idea  that  the  exposure  of  infected  articles  of  clothing 
and  bedding  in  the  sun  is  a  useful  sanitary  precaution  is  fully  sus- 
tained by  the  experimental  data  relating  to  the  action  of  heat,  desic- 
cation, and  sunlight. 

Electricity. — Cohn  and  Mendelssohn,  in  1879,  attempted  to  de- 
termine the  effect  of  the  galvanic  current  upon  bacteria.  Cultures 
were  placed  in  U  -tubes  through  which  a  constant  current  was  passed. 
A  feeble  current  was  found  to  be  without  effect.  A  strong  current 
from  two  elements,  maintained  for  twenty-four  hours,  restrained  de- 
velopment in  the  vicinity  of  the  positive  pole,  but  this  was  probably 
due  to  the  highly  acid  reaction  which  the  culture  liquid  acquired. 
When  a  current  from  five  elements  was  used  for  twenty-four  hours 
the  liquid  was  sterilized,  but  this  may  have  been  due  to  the  decided 
changes  produced  in  the  chemical  composition  of  the  culture  liquid 
rather  than  to  the  direct  action  of  the  galvanic  current. 

The  same  may  be  said  of  the  similar  results  obtained  in  later  ex- 
periments by  Apostoli  and  Laquerriere,  and  by  Prochownick  and 
Spaeth.  The  last-mentioned  investigators  found  that  the  positive  pole 
had  a  more  decided  effect  than  the  negative,  and  that  the  effect  de- 
pended upon  the  intensity  and  duration  of  the  current.  A  current  of 
fifty  milliamperes  passed  for  a  quarter  of  an  hour  did  not  kill  Staphy- 
lococcus  pyogenes  aureus,  but  a  current  of  sixty  milliamperes  main- 
tained for  the  same  time  did.  The  spores  of  Bacillus  anthracis 
required  a  current  of  two  hundred  to  two  hundred  and  thirty  milli- 
amperes during  an  hour  or  two.  In  these  experiments  the  cultures 
in  gelatin  were  attached  to  the  strips  of  platinum  serving  as  the  two 
poles,  and  these  were  immersed  in  a  solution  of  sodium  chloride.  As 
chlorine  was  disengaged  at  the  positive  pole,  the  germicidal  action  is 
attributed  to  this  gas  rather  than  to  the  direct  action  of  the  current 
upon  the  living  microorganisms. 

The  more  recent  researches  of  Spilker  and  Gottstein,  made  with 
an  induction  current  from  a  dynamo  machine,  are  more  valuable  in 


INFLUENCE   OF   PHYSICAL   AGENTS.  103 

estimating  the  power  of  this  agent  to  destroy  the  vitality  of  bacteria. 
The  current  was  passed  through  a  spiral  wire  which  was  wrapped 
around  a  test  tube  of  glass,  containing  the  microorganism  to  be  tested, 
suspended  in  distilled  water.  In  a  first  experiment  Bacillus  prodigi- 
osus,  suspended  in  sterilized  distilled  water  and  contained  in  test 
tubes  having  a  capacity  of  two  hundred  and  fifty  cubic  centimetres, 
was  subjected  to  a  current  having  an  energy  of  2.5  amperes  X  1.25 
volts  for  twenty-four  hours.  The  temperature  did  not  go  above 
30°  C.  No  development  occurred  when  the  microorganism  tested 
was  subsequently  planted  in  nutrient  gelatin.  Further  experiments 
gave  a  similar  result.  It  was  found  that  stronger  currents  were 
effective  in  shorter  time ;  but  in  no  case  was  sterilization  effected  in 
less  than  an  hour. 

Pressure. — D'Arsonval  and  Charrin  (1894)  submitted  a  culture 
of  Bacillus  pyocyaneus  tc  a  pressure  of  fifty  atmospheres,  under  car- 
bon dioxide.  At  the  end  of  four  hours  cultures  could  still  be  ob- 
tained, but  the  bacillus  had  lost  its  power  of  pigment  production.  A 
few  colonies  were  developed  after  six  hours'  exposure  to  this  pressure; 
but  after  twenty-four  hours  no  development  occurred. 

Agitation. — Meltzer  (1894)  has  shown  that  the  vitality  of  bacteria 
is  destroyed  by  protracted  and  violent  shaking,  which  causes  a  molec- 
ular disintegration  of  the  cells. 


VII. 
ANTISEPTICS  AND   DISINFECTANTS. 

GENERAL  ACCOUNT  OF  THE  ACTION  OF. 

THE  term  antiseptic  is  used  by  some  authors  to  designate  an 
agent  which  destroys  the  vitality  of  the  microorganisms  which  pro- 
duce septic  decomposition,  and  others  of  the  same  class.  We  prefer 
to  restrict  the  use  of  the  term  to  those  agents  which  restrain  the  de- 
velopment of  such  microorganisms  without  destroying  their  vitality. 
The  complete  destruction  of  vitality  is  effected  by  germicides  or  dis- 
infectants. Material  containing  the  germs  of  infectious  diseases  is 
infectious  material,  and  we  disinfect  it  by  the  use  of  agents  which 
destroy  the  living  disease  germs  or  pathogenic  bacteria  which  give 
it  its  infecting  power.  Such  an  agent  is  a  disinfectant.  But  we  ex- 
tend the  use  of  this  term  to  germicides  in  general — that  is,  to  those 
agents  which  kill  non-pathogenic  bacteria  as  well  as  to  those  which 
destroy  disease  germs.  All  disinfectants  are  also  antiseptics,  for 
agents  which  destroy  the  'vitality  of  the  bacteria  of  putrefaction  ar- 
rest the  putrefactive  process  ;  and  these  agents,  in  less  amount  than 
is  required  to  completely  destroy  vitality,  arrest  growth  and  thus 
act  as  antiseptics.  But  all  antiseptics  are  not  germicides.  Thus  a 
concentrated  solution  of  salt  or  of  sugar  will  prevent  the  putrefac- 
tive decomposition  of  organic  material,  animal  or  vegetable  ;  but  these 
agents  do  not  destroy  the  vitality  of  the  germs  of  putrefaction.  In 
a  certain  degree  of  concentration  they  are  antiseptics  and  are  largely 
used  for  the  preservation  of  meats  and  vegetables.  In  the  same  way 
many  mineral  salts  in  solutions  of  various  strengths  act  as  antisep- 
tics, and  some  of  these  in  still  stronger  solutions  are  disinfectants. 
Thus  mercuric  chloride,  when  introduced  into  a  culture  solution  in 
the  proportion  of  1  :  300,000,  will  restrain  the  development  of  anthrax 
spores,  but  to  insure  the  destruction  of  these  spores  a  solution  of 
1  : 1,000  must  be  used.  As  a  rule,  the  difference  between  restraining 
action — antiseptic — and  germicidal  power — disinfectant — is  not  so 
great  as  this.  We  give  below  some  recent  determinations  by  Boer 
which  illustrate  this  point,  the  test  organism  being  the  bacillus  of 
typhoid  fever  in  a  culture  in  bouillon  twenty-four  hours  old  : 


ANTISEPTICS   AXD    DISINFECTANTS.  165 


Restrains. 

Kills. 

Hydrochloric  acid  

1  :2100 

1  -300 

Sulphuric  acid  

1  :  1550 

1  :  500 

Silver  nitrate  

1  :  50000 

1  :  4000 

Sodium  arscniate  

1  :  6000 

1  :250 

Carbolic  acid  

1  :400 

1  :200 

Method  of  Determining  Antiseptic  rahte. — To  determine  the 
restraining  or  antiseptic  power  of  an  agent  for  a  particular  micro- 
organism, the  agent  is  dissolved  in  a  definite  proportion  in  a  suitable 
culture  medium,  which  is  then  inoculated  with  a  pure  culture  of  the 
test  organism  and  placed  in  favorable  circumstances — as  to  tempera- 
ture— for  its  growth.  At  the  same  time  a  control  experiment  is 
made  by  placing  another  portion  of  the  same  culture  medium,  inocu- 
lated with  the  same  microorganism,  in  the  same  conditions,  but  with- 
out the  addition  of  the  antiseptic  agent.  If  development  occurs  in 
the  control  experiment  and  not  in  the  culture  medium  containing 
the  antiseptic,  the  failure  to  grow  must  be  attributed  to  the  presence 
of  this  agent.  Having  made  a  preliminary  experiment,  we  are 
guided  by  the  result  in  further  experiments  to  determine  the  exact 
amount  required  to  restrain  development  under  the  same  conditions. 
Or  we  may  make  a  series  of  experiments  in  the  first  instance.  The 
problem  being,  for  example,  to  determine  the  antiseptic  value  of 
carbolic  acid  for  the  typhoid  bacillus,  we  may  add  this  agent  to  a 
definite  amount  of  bouillon  in  test  tubes  in  the  proportion  of  1  : 100, 
1 : 200,  1  : 300,  1  : 400,  1  :  500.  In  experiments  with  volatile  agents 
the  bouillon,  in  test  tubes  or  small  flasks,  must  be  sterilized  in  ad- 
vance, and  the  antiseptic  agent  introduced  by  means  of  a  sterilized 
pipette  with  great  care  to  prevent  the  accidental  contamination  of 
the  nutrient  medium.  In  experiments  with  non-volatile  agents  it  will 
be  best  to  sterilize  the  culture  medium  after  the  antiseptic  has  been 
added.  Next  we  inoculate  the  liquid  in  each  flask  with  a  pure  cul- 
ture of  the  test  organism.  The  flasks  are  then  placed  in  an  incubat- 
ing oven  at  35°  to  37°  C.  At  the  same  time  a  control,  not  containing 
any  carbolic  acid,  is  placed  in  the  oven.  At  the  end  of  twenty-four 
hours  the  control  will  be  found  to  be  clouded,  showing  an  abundant 
multiplication  of  the  bacillus.  Taking  the  result  of  Boer  above  given, 
we  would  expect  to  find  all  of  the  solutions  clear  except  that  contain- 
ing 1  :  500.  This  too  might  remain  clear  for  some  days  and  finally 
"  break  down,"  for  experience  shows  that  when  we  pass  the  point  at 
which  a  permanent  restraining  influence  is  exerted  there  may  be  a 
temporary  restraint  or  retardation  of  development.  For  this  reason 
we  must  continue  the  experiment  for  a  considerable  time — not  less 


1GG  ANTISEPTICS   AND    DISINFECTANTS. 

than  two  weeks.  Having  found  that  1  : 400  and  below  prevents 
development,  and  1  ;  500  does  not,  we  may  make  further  experiments 
to  determine  the  antiseptic  power  within  narrower  limits  ;  but  this 
is  hardly  necessary  from  a  practical  point  of  view. 

In  these  experiments  the  result  will  be  influenced  by  several  cir- 
cumstances, as  follows  : 

(a)  By  the  composition  of  the  nutrient  medium.     This  is  a 
very  important  factor,  especially  in  determining  the  antiseptic  value 
of  certain  metallic  salts.     The  presence  of  a  considerable  quantity 
of  albumin,  for  example,  reduces  greatly  the   antiseptic  power  of 
mercuric  chloride,  silver  nitrate,  creolin,  etc.    The  presence  of  a  sub- 
stance chemically  incompatible,  as,  for  example,  sodium  chloride  in 
testing  nitrate  of  silver,  will  of  course  neutralize  antiseptic  action. 

(b)  The  nature  of  the  test  organism.     Within  certain  limits  an 
antiseptic  for  one  microorganism  of  this  class  restrains  the  devel- 
opment of  all,  but  there  are  wide  differences  in  the  ability  of  differ- 
ent species  to  grow  in  the  presence  of  different  chemical  agents. 
Some  grow  readily  in  the  presence  of  a  considerable  amount  of  free 
acid,  others  are  restrained  by  a  slightly  acid  reaction  of  the  medium 
in  which  they  are  placed.     The  Bacillus  acidi  lactici,  for  example, 
can  thrive  in  the  presence  of  a  considerable  amount  of  the  acid 
which  is  a  product  of  its  growth,  but  there  is  a  limit  to  its  power  of 
developing  in  the  presence  of  this  and  other  acids.     So,  too,   Mi- 
crococcus  urese,  which  causes  the  alkaline  fermentation  of  urine, 
grows  in  the  presence  of  a  considerable  amount  of  carbonate  of  am- 
monia, but  is  finally  restrained  in  its  growth  by  this  alkaline  salt. 
The  following  determinations  by  Boer  show  the  difference  in  the 
antiseptic  power  of  hydrochloric  acid  for  certain  pathogenic  bacte- 
ria :  Bacillus  of  anthrax  (without  spores),  1  :  3,400  ;  diphtheria  bacil- 
lus, 1  :3,400  ;  glanders  bacillus,   1  :  700  ;  typhoid  bacillus,   1  :  2,100  ; 
cholera  spirillum,  1  :5,500.     It  will  be  noted  that  the  cholera  spiril- 
lum is  restrained  in  its  growth  by  about  one-eighth  the  amount  of 
hydrochloric  acid  which  is  required  to  prevent  the  development  of 
the  bacillus  of  glanders.     The  typhoid  bacillus  has  a  special  tole- 
rance for  carbolic  acid,  etc. 

(c)  The  temperature  at  which  the  experiment  is  made.     At 
the  temperature  most  favorable  for  growth  a  greater  proportion  of 
the  antiseptic  agent  is  required  than  at  unfavorable  temperatures- 
lower  or  higher. 

(d)  The  restraining  influence  for  spores  is  much  greater  than 
for  the  vegetative  form  of  bacteria. 

Methods  of  Determining  Germicide  Value. — The  disinfecting 
power  of  a  chemical  agent  is  determined  by  allowing  it  to  act  for  a 
given  time,  in  a  definite  proportion,  on  a  pure  culture  of  a  given 


ANTISEPTICS   AND   DISINFECTANTS.  167 

microorganism,  and  then  testing  the  question  of  loss  of  vitality  by 
culture  experiments  or  by  inoculations  of  infectious  disease  germs 
into  susceptible  animals. 

The  test  by  cultivation  is  the  most  reliable,  but  in  making  it 
several  points  must  be  kept  in  view.  Naturally  the  conditions  must 
be  such  as  are  favorable  for  the  growth  of  the  particular  microor- 
ganism which  serves  as  the  test ;  and  we  must  allow  a  considerable 
time  for  the  development  of  the  test  organism,  for  it  often  happens 
that  its  vital  activity  has  been  weakened  without  being  completely 
destroyed,  and  that  growth  will  occur  after  an  interval  of  several 
days,  while  in  the  control  experiment  it  has  perhaps  been  seen  at 
the  end  of  twenty-four  hours.  Another  most  important  point  is  the 
fact  that  some  of  the  disinfecting  agent  is  necessarily  carried  over 
with  the  test  organisms  when  these  are  transferred  to  a  nutrient 
medium  to  ascertain  whether  they  will  grow,  and  this  may  be  in  , 
sufficient  amount  to  restrain  their  development  and  lead  to  the  mis- 
taken inference  that  they  have  been  killed.  This  is  especially  true 
of  mercuric  chloride,  which  restrains  the  development  of  spores  in 
very  minute  amounts.  Spores  which  have  been  subjected  to  its  ac- 
tion in  comparatively  strong  solutions,  when  transferred  to  a  culture 
medium  may  fail  to  grow  because  of  the  restraining  influence  of 
the  mercuric  chloride  carried  over  at  the  same  time.  For  this  rea- 
son liquid  cultures  are  to  be  preferred  in  experiments  of  this  kind. 
When  the  test  organisms  are  planted  in  a  solid  culture  medium  the 
chemical  agent  is  left  associated  with  them  ;  in  a  liquid  culture,  on 
the  other  hand,  it  is  diluted,  and  the  microorganisms,  being  distri- 
buted through  the  nutrient  medium,  have  the  disinfecting  agent 
washed  from  their  surface.  In  the  case  of  mercuric  chloride,  how- 
ever, the  experiments  of  Geppert  show  that  the  agent  is  so  attached 
to  spores  which  have  been  subjected  to  its  action  that  ordinary 
washing  does  not  suffice.  Moreover,  spores  which  have  been  ex- 
posed to  the  action  of  mercuric  chloride  without  being  killed  are  re- 
strained in  their  growth  by  a  much  smaller  proportion  of  the  corro- 
sive sublimate  than  is  required  for  spores  not  so  exposed — according 
to  Geppert,  by  1  part  in  2,000,000.  Geppert  therefore  proposes,  in 
experiments  with  this  agent,  to  neutralize  the  mercuric  chloride 
which  remains  attached  to  the  test  organisms  by  washing  these  in 
a  solution  of  ammonium  sulphide,  by  which  the  sublimate  is  preci- 
pitated as  an  inert  sulphide. 

With  most  agents  simple  dilution  will  serve  the  purpose  of  pre- 
venting an  erroneous  inference  from  the  restraining  influence  of  the 
chemical  agent  being  tested.  If  we  carry,  by  means  of  a  platinum 
loop,  one  or  two  ose  into  five  to  ten  cubic  centimetres  of  bouillon, 
the  dilution  will  usually  be  beyond  the  restraining  influence  of  the 


108  ANTISEPTICS   AND   DISINFECTANTS. 

germicidal  agent ;  but  we  may  carry  the  dilution  still  further,  to  be 
on  the  side  of  safety,  by  inoculating  a  second  tube  containing  the 
same  amount  of  sterile  bouillon  from  the  first,  carrying  over  in  the 
same  way  one  or  two  ose.  We  will  still  be  very  sure  to  have  a 
considerable  number  of  the  microorganisms  to  test  the  question  of 
the  destruction  of  vitality.  Instead  of  bouillon  we  may  use  liquefied 
flesh-peptone-gelatin,  which  gives  us  the  same  advantage  as  to  dilu- 
tion of  the  disinfecting  agent ;  and  after  inoculating  two  tubes  as 
above  indicated,  we  may  make  Esmarch  roll  tubes  by  turning  them 
upon  a  block  of  ice.  The  development  of  colonies  will  show  that 
there  was  a  failure  to  disinfect ;  their  absence,  after  a  proper  inter- 
val, will  be  evidence  of  the  germicidal  action  of  the  agent  employed. 

Koch's  Method. — In  1881  Koch  published  his  extended  experi- 
ments made  to  determine  the  germicidal  power  of  various  chemical 
agents  as  tested  upon  anthrax  spores.  His  method  consisted  in  ex- 
posing silk  threads,  to  which  the  dried  spores  were  attached,  in  a 
solution  of  the  disinfecting  agent,  and  at  intervals  transferring  one 
of  these  threads  to  a  solid  culture  medium.  The  precaution  was 
taken  to  wash  the  thread  in  distilled  water  when  the  agent  tested  was 
supposed  to  be  likely  to  restrain  development.  In  these  experiments 
a  standard  solution  of  the  disinfecting  agent  was  used,  and  the  time 
of  exposure  was  varied  from  a  few  hours  to  many  days. 

The  Writer's  Method. — In  the  writer's  experiments,  made  in 
1880  and  subsequently,  a  different  method  has  been  adopted.  The 
time  has  been  constant — usually  two  hours — and  the  object  has  been 
to  find  the  minimum  amount  of  various  chemical  agents  which 
would  destroy  the  test  organisms  in  this  time ;  and  instead  of  sub- 
jecting a  few  of  the  test  organisms  attached  to  a  silk  thread  to  the 
action  of  the  disinfecting  agent,  a  certain  quantity  of  a  recent  cul- 
ture— usually  five  cubic  centimetres — has  been  mixed  with  an  equal 
quantity  of  a  standard  solution  of  the  germicidal  agent.  Thus  five 
cubic  centimetres  of  a  1  : 200  solution  of  carbolic  acid  would  be 
added  to  five  cubic  centimetres  of  a  recent  culture  of  the  typhoid 
bacillus,  for  example,  and  after  two  hours'  contact  one  or  two  ose 
would  be  introduced  into  a  suitable  nutrient  medium  to  test  the 
question  of  disinfection.  In  the  case  given  the  result  obtained 
would  be  set  down  as  the  action  of  a  solution  of  carbolic  acid  in  the 
proportion  of  1  :  400,  for  the  1  :  200  solution  was  diluted  by  the  addi- 
tion of  an  equal  quantity  of  the  culture. 

Other  experimenters  have  adopted  still  a  different  method.  In- 
stead of  using  a  considerable  and  definite  quantity  of  a  culture  con- 
taining the  test  organism,  they  introduce  one  or  two  ose  from  such 
a  culture  into  a  solution  containing  a  given  proportion  of  the  disin- 
fectant ;  then  after  exposure  for  a  given  time  the  nutrient  medium  is 
inoculated. 


ANTISEPTICS   AND    DISINFECTANTS. 


169 


These  different  methods  give  results  which  cannot  be  directly 
compared  one  with  another,  for  to  obtain  corresponding  results  we 
must  have  identical  conditions. 

Test  by  Inoculation  into  Susceptible  Animals. — In  testing  the 
action  of  disinfectants  upon  anthrax  spores  and  other  infectious  dis- 
ease germs,  we  may  inoculate  the  microorganisms,  after  exposure  to 
the  disinfectant,  into  a  susceptible  animal.  This  method  was  adopted 
by  the  writer  in  a  series  of  experiments  in  1881,  but  he  has  not  since 
employed  it,  for  reasons  set  forth  in  his  paper  giving  an  account  of 
these  experiments. 

"First.  The  test  organism  maybe  modified  as  regards  repro- 
ductive activity  without  being  killed ;  and  in  this  case  a  modified  form 
of  disease  may  result  from  the  inoculation,  of  so  mild  a  character  as 
to  escape  observation.  Second.  An  animal  which  has  suffered  this 
modified  form  of  the  disease  enjoys  protection,  more  or  less  perfect, 
from  future  attacks,  and  if  used  for  a  subsequent  experiment  may, 
by  its  immunity  from  the  effects  of  the  pathogenic  test  organism, 
give  rise  to  the  mistaken  assumption  that  this  had  been  destroyed 
by  the  action  of  the  germicidal  agent  to  which  it  had  been  sub- 
jected."1 

In  experiments  to  determine  the  value  of  an  agent  as  a  disinfec- 
tant, no  matter  by  what  method,  the  following  conditions,  which  in- 
fluence the  result,  should  be  kept  in  view  : 

(a)  The  difference  in  vital  resisting  power  of  different  species 
of  bacteria.  As  a  rule,  the  pathogenic  species  have  rather  less  re- 
sisting power  than  the  common  saprophytes,  and  the  micrococci 
have  greater  resisting  power  than  many  of  the  bacilli.  The  differ- 
ence in  the  vital  resisting  power  of  some  of  the  best  known  patho- 
genic species  is  shown  in  the  following  table,  which  we  have  made 
up  from  determinations  made  by  Boer — cultures  in  bouillon  twenty- 
four  hours  old  ;  time  of  exposure,  two  hours. 


Hydrochloric 
Acid. 

Caustic 
Soda. 

Chloride  of 
Gold  and 
Sodium. 

Nitrate 
of 
Silver. 

Carbolic 
Acid. 

Anthrax  bacillus  

1  :  1100 

1  :  450 

1  :8000 

1  :  20000 

1:300 

Diphtheria  bacillus  
Glanders  bacillus    

1:700 
1  :200 

1:300 
1  :150 

1  :  1000 
1  :400 

1  :2oOO 
1  :  4000 

1:300 
1  :300 

Typhoid  bacillus  

1  :300 

1:190 

1  :500 

1  :  4000 

1  :200 

Cholera  spirillum  

1  :  1350 

1  :150 

1  :  1000 

1  :4000 

1  :400 

(b)  The  presence  or  absence  of  spores.  The  reproductive  ele- 
ments known  as  spores  have  a  far  greater  resisting  power  to  chemi- 
cal agents,  as  well  as  to  heat,  than  have  the  vegetative  cells.  In 

1  Quoted  from  article  on  "  Germicides  and  Disinfectants/'  in  "  Bacteria,"  p.  212. 


170  ANTISEPTICS   AND   DISINFECTANTS. 

practical  disinfection,  therefore,  it  is  important  to  know  what  disease 
germs  form  spores  and  what  do  not.  The  following  are  known  to 
form  spores  :  The  bacillus  of  anthrax,  the  bacillus  of  tetanus,  the 
bacillus  of  malignant  oedema,  the  bacillus  of  symptomatic  anthrax, 
the  bacillus  of  foul  brood  (infectious  disease  of  bees).  The  following, 
so  far  as  is  known,  do  not  form  spores  :  The  pus  cocci  (Staphylo- 
coccus  pyogenes  albus,  aureus,  and  citreus,  and  Streptococcus  pyo- 
genes),  the  micrococcus  of  pneumonia,  the  bacillus  of  typhoid  fever, 
the  bacillus  of  glanders,  the  bacillus  of  diphtheria,  the  spirillum  of 
cholera,  the  spirillum  of  relapsing  fever. 

Many  agents  which  kill  the  growing  bacteria  are  incapable  of 
destroying  the  vitality  of  spores,  and  others  only  do  so  in  much 
stronger  solutions  or  after  a  long  exposure  to  their  action. 

(c)  The  number  of  bacteria  to  be  destroyed.     This  is  an  essen- 
tial factor  which  has  often  been  overlooked  by  those  making  experi- 
ments.   To  destroy  the  bacteria  carried  over  to  five  cubic  centimetres 
of  distilled  water  by  means  of  a  platinum  loop,  is  a  very  different 
matter  from  destroying  the  immensely  greater  number  in  five  cubic 
centimetres  of  a  recent  bouillon  culture. 

(d)  The  nature  and  quantity  of  associated  material.     The 
oxidizing  disinfectants,  like  permanganate  of  potash  and  chloride  of 
lime,  not  only  act  upon  the  bacteria,  destroying  them  by  oxidation, 
but  upon  all  organic  matter  with  which  they  come  in  contact,  and  at 
the  same  time  the  disinfecting  agent  is  destroyed  in  the  chemical 
reaction,  which  is  a  quantitative  one.     The  presence,  therefore,  of 
organic  material  in  association  with  the  bacteria  is  an  important 
factor,  and  if  this  is  in  excess  the  disinfectant  may  be  neutralized 
before  the  living  bacteria  are  destroyed.     Other  substances  which 
precipitate  the  disinfecting  agent  in  an  insoluble  form,  or  decompose 
it,  must  of  course  have  the  same  effect.    Thus  the  presence  of  sodium 
chloride  in  a  culture  medium  would  be  an  important  circumstance  if 
nitrate  of  silver  was  the  agent  being  tested,  as  the  insoluble  chloride 
would  be  precipitated.     And  in  the  case  of  mercuric  chloride  and 
certain  other  metallic  salts  the  presence  of  albumin  very  materially 
influences  the  result.     Van  Ermengem  states  that  the  cholera  spiril- 
lum in  bouillon  is  destroyed  in  half  an  hour  by  mercuric  chloride  in 
the  proportion  of  1:  60,000,  while  in  blood  serum  1: 800  was  required 
to  destroy  it  in  the  same  time. 

(e)  The  time  of  exposure  is  also  an  important  factor.     Some 
agents  act  very  promptly,  while  others  require  a  considerable  time  to 
effect  the  destruction  of  bacteria  exposed  to  their  action.     Thus  a 
solution  of  chloride  of  lime  containing  0.12  per  cent  destroys  the 
typhoid  bacillus   and   the   cholera  spirillum   in   five   minutes,    and 
the  anthrax  bacillus  in  one  minute  (Mssen).     On  the  other  hand, 


ANTISEPTICS   AND   DISINFECTANTS.  171 

quicklime  (milk  of  lime)  requires  a  contact  of  several  hours  to  in- 
sure the  destruction  of  pathogenic  bacteria. 

(/)  The  temperature  at  ivhich  the  exposure  is  made  has  a 
material  influence  upon  the  result.  This  is  shown  by  the  experi- 
ments of  Henle  and  of  Nocht.  As  a  general  rule  germicidal  activ- 
ity increases  in  direct  proportion  to  the  increase  in  temperature  from 
20°  0.  upward. 

(g}  The  degree  of  dilution  of  the  disinfecting  agent  is  also  a 
matter  of  importance .  This  is  especially  true  of  solutions  of  acids 
and  alkalies.  When  a  silk  thread  to  which  bacteria  are  attached  is 
suspended  in  an  acid  solution  the  essential  point  is  the  degree  of 
acidity,  and  not  the  quantity  of  acid  in  the  entire  solution.  But  if  a 
solution  of  permanganate  of  potash,  or  any  other  active  oxidizing 
agent,  is  used,  the  principal  question  is  not  the  degree  of  dilution,  but 
the  amount  of  the  disinfecting  agent  present  in  the  solution  used.  A 
grain  of  potassium  permanganate  dissolved  in  two  fluidounces  of 
distilled  water  would  probably  kill  just  as  many  bacteria  as  if  it 
were  dissolved  in  half  a  fluidounce,  although  the  time  required  for 
disinfection  might  be  longer. 

From  what  has  been  said  it  is  evident  that  the  simple  statement 
that  a  certain  agent  is  a  germicide  in  a  certain  proportion  has  but 
little  scientific  value,  unless  we  are  made  acquainted  with  the  condi- 
tions under  which  its  germicidal  action  has  been  tested. 


VIII. 

ACTION   OF   GASES   AND   OF   THE   HALOID  ELEMENTS 

UPON  BACTERIA. 

Oxygen. — Free  oxygen  is  essential  for  the  development  of  a  large 
number  of  species  of  bacteria — aerobics  ;  and  it  completely  prevents 
the  growth  of  others — anaerobics.  Many  bacteria,  even  when  freely 
exposed  in  a  desiccated  condition  to  the  action  of  atmospheric  oxygen, 
retain  their  vitality  for  a  long  time.  The  gradual  loss  of  pathogenic 
power  which  Pasteur  has  shown  occurs  in  cultures  of  the  anthrax 
bacillus  and  the  micrococcus  of  fowl  cholera,  is  ascribed  by  him  to 
exposure  to  oxygen,  and  as  proof  of  this  he  states  that  cultures  kept 
in  hermetically  sealed  tubes  do  not  lose  their  virulence  in  the  same 
degree.  But  other  circumstances  may  influence  the  result.  Thus 
some  of  the  products  of  growth  which  accumulate  in  culture  fluids 
have  an  injurious  effect  upon  the  vitality  of  the  bacteria  which  pro- 
duced them,  and  in  time  may  cause  a  complete  destruction  of  vitality. 
In  cultures  exposed  to  the  air  these  products  would  be  in  a  more 
concentrated  solution  from  the  gradual  evaporation  of  the  culture 
liquid.  It  must  also  be  remembered  that  light  in  the  presence  of 
oxygen  is  a  germicidal  agent. 

The  experiments  of  Frankel  show  that  the  aerobic  bacteria  grow 
abundantly  in  the  presence  of  pure  oxygen,  and  some  species  even 
more  so  than  in  ordinary  air.  Micrococcus  prodigiosus,  however, 
appeared  to  be  unfavorably  affected  by  pure  oxygen,  inasmuch  as  it 
did  not  produce  pigment  so  readily  as  when  cultivated  in  ordinary  air. 

Nascent  oxygen  is  a  very  potent  germicidal  agent,  as  will  be  seen 
in  our  account  of  such  oxidizing  disinfectants  as  potassium  perman- 
ganate and  the  hypochlorite  of  lime. 

Ozone. — It  was  formerly  supposed  that  ozone  would  prove  to  be 
a  most  valuable  agent  for  disinfecting  purposes  ;  but  recent  experi- 
ments show  that  it  is  not  so  active  a  germicide  as  was  anticipated, 
and  that  from  a  practical  point  of  view  it  has  comparatively  little 
value. 

Lukaschewitsch  found  that  one  gramme  in  the  space  of  a  cubic 
metre  failed  to  kill  anthrax  spores  in  twenty-four  hours.  The  cholera 
spirillum  in  a  moist  state  was  killed  in  this  time  by  the  same  amount, 
but  fifteen  hours'  exposure  failed  to  destroy  it.  Ozone  for  these  ex- 
periments was  developed  by  means  of  electricity. 


ACTION   OF   GASES   AND    HALOID    ELEMENTS   UPON   BACTERIA.     173 

Wyssokowicz  found  that  the  presence  of  ozone  in  a  culture  me- 
dium restrained  the  development  of  the  anthrax  bacillus,  the  bacillus 
of  typhoid  fever,  and  others  tested,  but  concludes  that  this  is  rather 
due  to  the  oxidation  of  bases  contained  in  the  nutrient  medium  than 
to  a  direct  action  upon  the  pathogenic  bacteria. 

Sonntag,  in  his  carefully  conducted  experiments,  in  which  a  cur- 
rent of  ozonized  air  was  made  to  pass  over  silk  threads  to  which  were 
attached  anthrax  spores,  had  an  entirely  negative  result.  The  an- 
thrax bacillus  from  the  spleen  of  a  mouse,  and  free  from  spores,  was 
then  tested,  also  with  a  negative  result,  even  after  exposure  to  the 
ozonized  air  for  twenty  minutes  at  a  time  on  four  successive  days.  In 
another  experiment  several  test  organisms  (Bacillus  anthracis,  Bacil- 
lus pneumonise  of  Friedlander,  Staphylococcus  pyogenes  aureus, 
Staphylococcus  pyogenes  albus,  Bacillus  murisepticus,  Bacillus 
crassus  sputigenus)  were  exposed  on  silk  threads  for  twenty-four 
hours  in  an  atmosphere  containing  4. 1  milligrammes  of  ozone  to  the 
litre  of  air  (0. 19  volumes  per  cent).  The  result  was  entirely  negative. 
When  the  amount  was  increased  to  13.53  milligrammes  per  litre  the 
anthrax  bacillus  and  Staphylococcus  pyogenes  albus  failed  to  grow 
after  twenty-four  hours'  exposure.  The  conclusion  reached  by  Nis- 
sen,  from  his  own  experiments  and  a  careful  consideration  of  those 
previously  made  by  others,  is  that  ozone  is  of  no  practical  value  as  a 
germicide  in  therapeutics  or  disinfection. 

Hydrogen. — This  gas  has  no  injurious  effect  upon  bacteria,  as  is 
shown  by  the  fact  that  the  anaerobic  and  facultative  anaerobic  species 
grow  readily  in  an  atmosphere  of  pure  hydrogen. 

Hydrogen  peroxide  in  solution  in  water  is  a  valuable  antiseptic 
and  deodorant,  but  its  value  as  a  germicide  has  been  very  much 
overestimated.  Miquel,  in  his  experiments  to  determine  the  anti- 
septic value  of  various  agents,  places  H2O2  third  in  the  list  of  "  sub- 
stances eminently  antiseptic,"  and  states  that  it  prevents  the  develop- 
ment of  the  bacteria  of  putrefaction  in  the  proportion  of  1: 20,000. 

In  the  writer's  experiments  (1885)  a  solution  was  used  which 
contained  at  first  4. 8  per  cent  of  H2O2,  and  five  per  cent  of  sulphuric 
acid  which  was  added  by  the  chemist  who  prepared  the  solution,  to 
prevent  loss  of  the  hydrogen  peroxide.  At  the  end  of  a  month  the 
amount  of  H2O2  was  again  estimated,  and  found  to  be  3.98  per  cent. 
Five  weeks  later  the  proportion  was  2.4  per  cent.  Tested  upon 
^broken-down"  beef  tea,  this  solution  was  found  to  destroy  the 
vitality  of  the  bacteria  of  putrefaction  contained  in  it,  in  two  hours7 
time,  in  the  proportion  of  thirty  per  cent  (about  1.2  per  cent  of  H202). 
Anthrax  spores  were  killed  in  the  same  time  by  a  twenty-per-cent 
solution  (0.8  per  cent  H2O2).  Tested  upon  a  pure  culture  of  pus 
cocci,  it  was  active  in  the  proportion  of  ten  per  cent  (0.4  per  cent  of 


174  ACTION   OF   GASES   AND   OF   THE 

H2O2);  a  solution  containing  0.24  per  cent  of  H2O2  failed  to  kill  pus 
cocci.  But  the  solution  used  in  these  experiments  contained  also  five 
per  cent  of  sulphuric  acid,  which  by  itself  kills  niicrococci  in  the  pro- 
portion of  1 : 200.  My  conclusion  was  that,  unless  the  chemists  can 
furnish  more  concentrated  solutions  which  will  keep  better  than  that 
with  which  I  experimented,  we  are  not  likely  to  derive  any  practical 
benefit  from  the  use  of  hydrogen  peroxide  as  a  disinfectant. 

Altehof er  more  recently  has  experimented  with  a  solution  contain- 
ing 9.7  per  cent  of  H2O2,  and  reports  the  following  results:  He  added 
to  ninety-eight  cubic  centimetres  of  hydrant  water  two  cubic  centi- 
metres of  a  bouillon  culture  of  the  typhoid  bacillus,  and  to  this  was 
added  sufficient  of  his  aqueous  solution  of  H2O2  to  make  the  propor- 
tion present  1: 1,000.  At  the  end  of  twenty-four  hours  the  bacillus 
was  proved  by  culture  experiments  to  be  killed.  Water  containing 
the  cholera  spirillum,  treated  in  the  same  way,  was  not  entirely  steril- 
ized, as  a  few  colonies  developed  in  Esmarch  roll  tubes  ;  but  the  gen- 
eral result  of  his  experiments  was  that  the  ordinary  water  bacteria, 
and  the  pathogenic  bacteria  named  (cholera,  typhoid)  when  sus- 
pended in  water,  required  for  their  destruction  exposure  for  twenty- 
four  hours  in  a  solution  containing  one  part  of  H2O2  in  one  thousand 
of  water. 

Carbon  Dioxide. — The  experiments  of  Frankelshow  that  certain 
bacteria  grow  in  an  atmosphere  of  C03  as  well  as  in  the  air  ;  among 
these  are  the  bacillus  of  typhoid  fever  and  the  pneumonia  bacillus 
of  Friedlander.  Other  species  are  slightly  restricted  in  their  growth, 
e.g.  Bacillus  prodigiosus,  Proteus  vulgaris.  Still  others  grow  only 
when  the  temperature  is  elevated,  including  the  pus  cocci  and  the 
bacillus  of  swine  pest.  Most  of  the  saprophytic  bacteria  failed  to 
grow  in  an  atmosphere  of  CO2,  although  their  vitality  was  not  de- 
stroyed by  it.  Certain  pathogenic  species  were,  however,  killed  by 
the  action  of  this  gas,  among  others  the  cholera  spirillum,  Bacillus 
anthracis,  and  Staphylococcus  pyogenes  aureus. 

Leone  and  Hochstetter  had  previously  reported  that  certain  bac- 
teria are  injuriously  affected  by  CO2.  Frankel  also  found  that  the 
growth  of  strictly  anaerobic  species  was  restricted  in  an  atmosphere 
of  carbon  dioxide.  The  aerobic  species  which  failed  to  grow  in  pure 
CO2  grew  abundantly  when  a  little  atmospheric  oxygen  was  ad- 
mitted. In  the  experiments  of  Frankla  id  the  cholera  spirillum  and 
the  Finkler-Prior  spirillum  failed  to  develop  in  an  atmosphere  of 
CO2,  and  at  the  end  of  eight  days  were  no  longer  capable  of  growth 
when  the  carbon  dioxide  was  replaced  with  atmospheric  air. 

Carbonic  Oxide. — Frankland's  experiments  show  that  an  atmo- 
sphere of  this  gas  is  not  favorable  to  the  growth  of  the  cholera  spiril- 
lum or  of  the  Finkler-Prior  spirillum,  although  it  did  not  entirely 


HALOID   ELEMENTS   UPON   BACTERIA.  175 

prevent  development,  and  after  seven  days' exposure  the  spirilla  were 
not  all  killed,  although  a  comparatively  small  number  of  colonies  de- 
veloped. Bacillus  pyocyaneus  failed  to  grow  in  an  atmosphere  of 
CO,  but  when  air  was  admitted,  at  the  end  of  seven  or  eight  days, 
abundant  development  occurred. 

Methane,  CH4. — We  have  no  exact  experiments  to  determine 
the  action  of  marsh  gas  in  a  pure  state  on  bacteria,  but  the  experi- 
ments of  Kladakis  upon  illuminating  gas  maybe  taken  as  repre- 
senting approximately  what  might  be  expected  from  exposure  in 
pure  CH4.  An  analysis  of  the  gas  used  in  his  experiments  showed 
it  to  contain  37.97  per  cent  of  hydrogen,  39.37  per  cent  of  methane 
(CH4),  9.99  per  cent  of  nitrogen,  4.29  per  cent  of  ethene  (C2H4),  3.97 
per  cent  of  carbonic  oxide  (CO),  O.G1  per  cent  of  oxygen,  and  0.41  per 
cent  of  carbon  dioxide.  As  hydrogen  and  nitrogen  are  neutral,  and 
carbonic  oxide  is  shown  by  the  experiments  of  Frankland  not  to  act 
as  a  germicide  after  several  days'  exposure  to  its  action,  the  positive 
results  obtained  in  the  experiments  of  Kladakis  may  be  ascribed  to 
the  presence  of  CH4  (39.37  per  cent)  or  of  C2H4  (4.29  per  cent),  or  of 
both  together. 

A  large  number  of  microorganisms  were  tested,  and  among  these 
Proteus  vulgaris  alone  grew  in  an  atmosphere  of  illuminating  gas. 
The  others  not  only  failed  to  grow  in  such  an  atmosphere,  but  were 
destroyed  by  it.  Cultures  of  Bacillus  anthracis,  Staphylococcus  pyo- 
genes  aureus,  and  Spirillum  cholerse  Asiatics  were  sterilized  in  half 
an  hour  by  the  action  of  this  gas.  The  gas  was  also  found  to  be  un- 
suitable for  anaerobic  cultures. 

Xitrous  Oxide,  N2O. — The  experiments  of  Frankland,  made 
upon  the  cholera  spirillum,  the  spirillum  of  Finkler-Prior,  and  the 
bacillus  of  green  pus,  gave  results  similar  to  those  obtained  with  CO, 
viz. ,  seven  days'  exposure  in  an  atmosphere  of  this  gas  failed  to  de- 
stroy the  test  organisms,  but  completely  restrained  the  growth  of 
Bacillus  pyocyaneus  and  interfered  materially  with  the  development 
of  the  two  species  of  spirillum  without  entirely  preventing  it. 

Sitrogen  Dioxide,  NO. — Frankland  found  that  his  test  organ- 
isms were  quickly  killed  by  this  gas  (Bacillus  pyocyaneus,  Spirillum 
cholerse  Asiatics,  Spirillum  Finkler-Prior). 

Hydrosulphuric  Acid,  H2S. — In  the  experiments  of  Frankland 
'this  gas  proved  to  be  quickly  fatal  to  the  bacteria  tested  (Bacillus 
pyocyaneus,  Spirillum  cholera?  Asiatics,  Spirillum  Finkler-Prior). 
On  the  other  hand,  Grauer  found  that  this  gas  did  not  exercise  any 
injurious  influence  upon  the  tubercle  bacillus,  the  bacillus  of  anthrax, 
the  typhoid  bacillus,  or  the  cholera  spirillum,  after  the  exposure  of 
these  microorganisms  in  a  current  of  the  gas  for  an  hour. 

It  has  been  shown  by  the  experiments  of   Holschewnikoff   and 


176  ACTION   OF   GASES   AND   OF   THE 

others  that  certain  species  of  bacteria  cause  an  abundant  evolution 
of  H2S  as  a  result  of  their  development  in  an  albuminous  medium 
(Bacillus  sulfureus  and  Proteus  sulfureus). 

Sulphur  Dioxide,  SO2. — Very  numerous  experiments  have  been 
made  with  this  gas,  owing  to  the  fact  that  it  has  been  extensively 
used  in  various  parts  of  the  world  for  the  disinfection  of  hospitals, 
ships,  apartments,  clothing,  etc. 

In  the  writer's  experiments,  made  in  1880,  dry  vaccine  virus  on 
ivory  points  was  disinfected  by  exposure  for  twelve  hours  in  an  at- 
mosphere containing  one  volume  per  cent  of  this  gas,  and  liquid 
virus,  exposed  in  a  watch  glass,  by  one-third  of  this  amount.  Sub- 
sequent experiments  (1885)  showed  that  pus  micrococci  were  killed 
by  exposure  for  eighteen  hours  in  a  dry  atmosphere  containing  twenty 
volumes  per  cent  of  SO2,  but  that  four  volumes  per  cent  failed.  In 
the  presence  of  moisture  this  gas  has  considerably  greater  germicidal 
power  than  this,  owing,  no  doubt,  to  the  formation  of  the  more  ac- 
tive agent,  sulphurous  acid  (H2SO3).  But  in  a  pure  state  anhydrous 
sulphur  dioxide  does  not  destroy  spores.  The  writer  has  shown  that 
the  spores  of  Bacillus  anthracis  and  Bacillus  subtilis  are  not  killed  by 
contact  for  some  time  with  liquid  SO2  (liquefied  by  pressure).  Koch 
exposed  various  species  of  spore-bearing  bacilli  in  a  disinfection  cham- 
ber for  ninety-six  hours,  the  amount  of  SO2  at  the  outset  of  the  ex- 
periment being  6.13  volumes  per  cent,  and  at  the  end  3.3  per  cent. 
The  result  was  entirely  negative. 

But  in  the  absence  of  spores  the  anthrax  bacillus,  in  a  moist  con- 
dition, attached  to  silk  threads,  was  destroyed  in  thirty  minutes  in 
an  atmosphere  containing  one  volume  per  cent. 

In  another  of  Koch's  experiments  the  amount  of  SO2  in  the  disin- 
fection chamber  was  at  the  outset  0.84  per  cent,  and  at  the  end  of 
twenty-four  hours  0.55  per  cent.  An  exposure  of  one  hour  in  this  at- 
mosphere killed  anthrax  bacilli  attached  to  silk  threads,  in  a  moist 
.condition;  but  four  hours'  exposure  failed  to  kill  Bacillus  prouigiosus 
growing  on  potato,  while  twenty-four  hours'  exposure  was  successful. 
A  similar  result  was  obtained  with  Bacillus  pyocyaneus. 

Thinot,  as  a  result  of  experiments  made  in  1890,  arrives  at  the 
conclusion  that  the  specific  germs  of  tuberculosis,  glanders,  farcy  of 
cattle,  typhoid  fever,  cholera,  and  diphtheria  are  destroyed  by  twenty- 
four  hours'  exposure  in  an  atmosphere  containing  SO2  developed  by 
the  combustion  of  sixty  grains  of  sulphur  per  cubic  metre.  This 
amount  corresponds  closely  with  that  fixed  by  the  Committee  on  Dis- 
infectants of  the  American  Public  Health  Association  on  the  experi- 
mental evidence  obtained  by  the  writer  in  1885.  But  the  committee 
insisted  upon  the  presence  of  moisture  and  made  the  time  of  exposure 
twelve  hours — "exposure  for  twelve  hours  to  an  atmosphere  con- 


HALOID   ELEMENTS   UPON   BACTERIA.  177 

taining  at  least  four  volumes  per  cent  of  this  gas  in  the  presence  of 
moisture. " 

Chlorine. — The  haloid  elements  are  active  germicidal  agents, 
especially  chlorine  on  account  of  its  affinity  for  hydrogen,  and  the 
consequent  release  of  nascent  oxygen  when  it  comes  in  contact  with 
microorganisms  in  a  moist  condition.  And  for  the  same  reason  this 
agent  is  a  much  more  active  germicide  in  the  presence  of  moisture 
than  in  a  dry  condition.  The  experiments  of  Fischer  and  Proskauer 
showed  that  when  dried  anthrax  spores  were  exposed  for  an  hour  in 
an  atmosphere  containing  44. 7  per  cent  of  dry  chlorine  they  were  not 
destroyed  ;  but  if  the  spores  were  previously  moistened  and  were  ex- 
posed in  a  moist  atmosphere  for  the  same  time,  four  per  cent  was 
effective,  and  when  the  time  was  extended  to  three  hours  one  per 
cent  destroyed  their  vitality.  The  anthrax  bacillus,  in  the  absence  of 
spores,  was  killed  by  exposure  in  a  moist  atmosphere  containing  1 
part  to  2,500,  the  time  of  exposure  being  twenty-four  hours,  and  the 
same  amount  was  effective  for  Micrococcus  tetragenus  ;  the  strepto- 
coccus of  erysipelas  and  the  micrococcus  of  fowl  cholera  were  killed  in 
three  hours  by  1  : 2,500,  and  in  twenty-four  hours  by  1:  25,000.  The 
bacillus  of  mouse  septica3mia  and  the  tubercle  bacillus  were  killed  in 
one  hour  by  1  : 200. 

In  the  writer's  experiments  (1880)  four  children  were  vaccinated 
with  virus  from  ivory  points  which  had  been  exposed  for  six  hours  in 
an  atmosphere  containing  one-half  per  cent  of  chlorine ;  also  with 
four  points,  from  the  same  lot,  not  disinfected.  Vaccination  was  un- 
successful in  every  case  with  the  disinfected  points,  and  successful 
with  those  not  disinfected.  Koch  found  that  anthrax  spores  failed 
to  grow  after  twenty-four  hours'  exposure  in  chlorine  water.  In 
the  experiments  of  De  la  Croix  to  determine  the  antiseptic  power  of 
this  agent,  it  was  found  that  when  present  in  unboiled  beef  infusion 
in  the  proportion  of  1  : 15,600  no  development  of  bacteria  occurred. 
Miquel  gives  the  antiseptic  value  of  chlorine  as  1  :  4,000. 

Chloroform. — Immersion  for  one  hundred  days  in  chloroform 
does  not  destroy  the  vitality  of  anthrax  spores  (Koch).  This  agent 
is  without  effect  on  the  virus  of  symptomatic  anthrax"  (Arloing, 
Cornevin,  and  Thomas).  Salkowski  found  that  the  anthrax  bacillus 
in  the  absence  of  spores,  and  the  cholera  spirillum,  were  killed  by 
being  immersed  in  chloroform  water  for  half  an  hour.  Kirchner 
reports  still  more  favorable  results.  In  his  experiments  a  one-per- 
cent solution  killed  the  cholera  spirillum  in  less  than  a  minute,  and 
a  one-quarter-per-cent  solution  in  an  hour.  But  the  typhoid  bacillus 
required  at  least  one-half  per  cent  acting  for  an  hour. 

Iodine. — In  the  writer's  experiments  (1880)  iodine  in  aqueous 
solution  with  potassium  iodide  was  found  to  be  fatal  to  Micrococcus 
12 


178  ACTION   OF  GASES   AND    OF   THE 

pneumonise  crouposscin  the  proportion  of  1  : 1,000,  and  to  the  staphy- 
lococci  of  pus  in  1  :  500 — time  of  exposure  two  hours.  Iodine  water 
was  found  by  Koch  to  destroy  the  vitality  of  anthrax  spores  in 
twenty-four  hours,  but  a  two-per-cent  solution  in  alcohol  failed  to 
destroy  anthrax  spores  in  forty-eight  hours.  In  the  experiments  of 
Schill  and  Fischer  twenty  hours'  contact  with  a  solution  of  the 
strength  of  1  :  500  failed  to  destroy  the  virulence  of  tuberculous  spu- 
tum, as  tested  by  inoculation  experiments.  The  antiseptic  value  of 
iodine  is  given  by  Miquel  as  1  : 4,000. 

Bromine. — Fischer  and  Proskauer  have  studied  the  action  of 
bromine  vapor  upon  various  microorganisms.  They  found  that  ex- 
posure for  three  hours  in  a  dry  atmosphere  to  three  per  cent  does 
not  destroy  the  tubercle  bacillus  in  sputum  or  the  spores  of  an- 
thrax. But  when  the  atmosphere  is  saturated  with  moisture  1  :  500 
is  effective  •  and  when  the  time  of  exposure  was  extended  to  twenty- 
four  hours,  1  : 3,500.  A  two-per-cent  solution  destroys  the  vitality 
of  anthrax  spores  in  twenty-four  hours  (Koch).  Bromine  vapor  is 
an  active  agent  for  the  destruction  of  the  virus  of  symptomatic  an- 
thrax (Arloing,  Cornevin,  and  Thomas).  Miquel  gives  the  antisep- 
tic value  of  bromine  as  1  : 1,666,  which  is  considerably  below  that  of 
chlorine  and  iodine. 

Iodine  Trichloride. — According  to  Behririg,  we  possess  in  this 
agent  a  disinfectant  which  possesses  the  potency  of  free  chlorine  and 
iodine  without  having  their  disadvantages.  As  prepared  by  O.  Rie- 
del  it  is  a  yellowish-red  powder  of  penetrating  odor.  It  remains  un- 
changed for  weeks  in  concentrated  aqueous  solution  (five  per  cent). 
A  one-per-cent  solution  destroys  anthrax  spores  suspended  in  water 
almost  instantly,  and  a  0.2-per- cent  solution  within  a  few  minutes. 
Anthrax  spores  in  blood  serum  are  killed  by  a  one-per-cent  solution 
in  forty  minutes  (Behring).  Langenbuch  found  that  a  solution  of 
1  : 1,000  kills  spores  in  a  short  time,  and  that  when  added  to  nutri- 
ent gelatin  in  the  proportion  of  1  : 1,200  it  restrains  the  develop- 
ment of  bacteria. 

lodoform. — Numerous  experiments  have  been  made  with  this 
agent,  which  show  that  it  has  little,  if  any,  germicidal  power  ;  but 
it  acts  to  some  extent  as  an  antiseptic.  Tilanus  reports  that  the  tu- 
bercle bacillus  will  not  grow  in  glycerin-agar  cultures  to  which  a 
small  quantity  of  iodof orm  has  been  added,  and  that  a  pure  culture 
of  the  tubercle  bacillus  was  not  killed  in  six  days  by  exposure  to 
iodof  orm  vapor,  but  that  after  six  weeks'  exposure  it  failed  to  grow. 
The  experiments  of  Neisser  and  of  Buchner  show  that  while  most 
bacteria  are  not  injuriously  affected  by  exposure  to  iodof  orm  vapor, 
the  cholera  spirillum  and  the  Finkler-Prior  spirillum  are  restrained  in 
their  growth  by  such  exposure.  When  plate  cultures  of  the  cholera 


HALOID    ELEMENTS   UPON   BACTERIA.  179 

spirillum  were  placed  under  a  bell  jar  beside  iodoform  powder 
no  development  occurred,  but  when  they  were  removed  colonies  de- 
veloped, showing  that  the  spirilla  were  not  killed. 

Iodoform  Ether,  according  to  Yersin,  is  fatal  to  the  tubercle  ba- 
cillus in  one-per-cent  solution  in  five  minutes.  Cadeac  and  Meunier 
found  that  a  saturated  solution  required  thirty-six  hours  to  kill  the 
bacillus  of  typhoid  fever. 

loclol. — In  experiments  made  by  the  writer  (1885)  this  agent  was 
found  to  be  without  germicidal  power.  Riedlin  found  it  without  any 
action,  even  upon  the  cholera  spirillum. 

Hydrofluoric  Acid,  HF1. — From  a  series  of  experiments  made 
with  this  gas,  Grancher  and  Chautard  arrive  at  the  conclusion  that 
"  the  direct  and  prolonged  action  of  hydrofluoric  acid  upon  the  tuber- 
cle bacillus  diminishes  its  virulence  but  does  not  kill  it." 

Sozoiodol  Acid, according  to  Draer,is  a  phenol,  in  which  two  atoms 
of  hydrogen  are  replaced  by  two  of  iodine  arid  one  atom  by  the  group 
HSO3.  This  acid  and  its  salts  with  soda,  potash,  zinc,  and  mercury 
have  been  tested  by  the  author  named.  The  acid  and  its  salt  with 
mercury  were  found  to  destroy  the  cholera  spirillum  in  two  hours'  time 
in  two-per-cent  solution.  A  two-per-cent  solution  of  phenol  would 
have  accomplished  the  same  result  and  in  less  time.  Tribromphenol, 
according  to  Draer,  is  less  active  than  sozoiodol  acid ;  and  it  appears 
from  the  experimental  evidence  on  record  that  combinations  of 
iodine,  chlorine,  or  bromine  with  phenol  are  less  active  that  the 
haloid  elements  alone.  According  to  Karpow  (1893)  monochlor- 
phenol,  tested  upon  anthrax  spores  attached  to  silk  threads,  proved 
to  be  decidedly  more  active  than  phenol. 

Nosophen  (tetraiodphenolphthalein),  according  to  Lieveii  (1895) 
contains  sixty-one  per  cent  of  iodine.  It  is  entirely  insoluble  in 
water.  When  added  to  nutrient  gelatin  in  the  proportion  of  one- 
quarter  per  cent  it  prevented  the  development  of  the  anthrax  bacillus 
and  of  Staphylococcus  aureus,  but  failed  to  prevent  the  development 
of  Bacillus  pyocyaneus  (Lieven). 


IX. 


ACTION   OF   ACIDS   AND   ALKALIES. 

Sulphuric  Acid,  H2SO4. — The  experiments  of  Koch  (1881) 
showed  that  anthrax  spores  were  still  capable  of  growing  after  ex- 
posure in  a  one-per-cent  solution  of  sulphuric  acid  for  twenty  days. 
In  the  writer's  experiments  (1885)  a  four-per-cent  solution  failed  to 
destroy  the  spores  of  Bacillus  subtilis  in  four  hours,  and  an  eight- 
per-cent  solution  was  found  to  be  required  for  the  sterilization  of 
culture  fluids  containing  spores  ;  but  the  multiplication  of  the  bacte- 
ria of  putrefaction  was  prevented  by  the  presence  of  this  acid  in  a 
culture  solution  in  the  proportion  of  1  :  800.  Pus  micrococci  were 
destroyed  by  exposure  for  two  hours  in  a  solution  containing  1  :  200. 

The  experiments  of  Boer  show  that  there  is  a  considerable  differ- 
ence in  the  resisting  power  of  different  pathogenic  bacteria.  The 
time  of  exposure  being  two  hours,  cultures  in  bouillon  twenty-four 
hours  old  gave  the  following  results  : 


Restrains 
development. 

Destroys 
vitality. 

1  :  2550 

1  :  1300 

Diphtheria  bacillus  

1  :  2050 

1  :500 

Glanders  bacillus  

1  :750 

1  :200 

Typhoid  bacillus  

1  :  1550 

1  :500 

Cholera  spirillum  

1  :  7000 

1  :  1300 

Leitz,  in  his  studies  relating  to  the  bacillus  of  typhoid  fever, 
reports  the  following  results  :  The  dejections  of  typhoid  patients, 
mixed  with  an  equal  proportion  of  the  disinfecting  solution,  were 
sterilized  by  a  five-per-cent  solution  of  sulphuric  acid  in  three  days. 
A  pure  culture  was  sterilized  in  fifteen  minutes  by  two  per  cent,  and 
in  five  minutes  by  five  per  cent. 

Sulphurous  Acid,  H2SO3. — In  the  writer's  experiments  (1885) 
micrococci  were  destroyed  in  two  hours  by  1  :  2,000  by  weight  of  SO, 
added  to  water.  Kitasato  found  that  solutions  of  sulphurous  acid 
in  the  proportion  of  0.28  per  cent  killed  the  typhoid  bacillus,  and 
0. 148  per  cent  the  cholera  spirillum.  De  la  Croix  found  that  one 


ACTION   OF   ACIDS   AND    ALKALIES.  181 

gramme  of  SO2  added  to  two  thousand  of  bouillon  prevents  the  de- 
velopment of  putrefactive  bacteria  and  after  a  time  destroys  the 
vitality  of  these  bacteria.  The  writer  found  that  pus  cocci  failed  to 
grow  in  a  culture  solution  containing  one  part  of  SO2  in  five  thousand 
of  water. 

Nitric  Acid,  HNO3. — In  the  writer's  experiments  an  eight-per- 
cent solution  which  contained  0.819  gramme  of  HNO3  in  each  cubic 
centimetre  sterilized  broken-down  beef  tea  containing  spores,  and 
five  per  cent  failed  to  do  so.  Kitasato,  in  experiments  upon  the  chol- 
era spirillum  and  typhoid  bacillus,  obtained  results  corresponding 
with  those  obtained  with  hydrochloric  acid — 0.2  per  cent  destroyed 
vitality  at  the  end  of  four  or  five  hours.  In  these  experiments  the 
acid  used  contained  0.35  gramme  HNO3  in  one  cubic  centimetre. 

Nitrous  Acid. — In  the  writer's  experiments  on  vaccine  virus  (1880) 
exposure  for  six  hours  in  an  atmosphere  containing  one  per  cent  of 
nitrous  acid  destroyed  the  virulence  of  dried  virus  upon  ivory  points. 

Hydrochloric  Acid,  HC1. — Anthrax  spores  are  destroyed  in  ten 
days  by  a  two-per-cent  solution,  but  not  in  five  days  (Koch).  Tested 
upon  broken-down  beef  tea  containing  spores  of  Bacillus  subtilis,  it 
was  effective  in  two  hours  in  the  proportion  of  fifteen  per  cent,  but 
failed  in  ten  per  cent  (Sternberg).  In  the  experiments  of  Kitasato  this 
acid  destroyed  the  typhoid  bacillus  in  five  hours  in  the  proportion  of 
0.2  per  cent,  and  the  cholera  spirillum  in  0.132  per  cent — the  acid 
used  contained  0. 26  gramme  HC1  in  one  cubic  centimetre.  We  give 
the  more  recent  determinations  of  Boer  in  tabular  form.  Its  germi- 
cidal  power  was  tested  upon  bouillon  cultures  which  had  been  kept 
for  twenty-four  hours  in  an  incubating  oven  ;  time  of  exposure  to 
the  action  of  the  acid  solution,  two  hours. 


Restrains 
development. 

Destroys 
vitality. 

Anthrax  bacillus  

1  :3400 

1  :  1100 

Diphtheria  bacillus  

1  :  3400 

1  :700 

Glanders  bacillus  *  

1  :  700  ' 

1  -200 

Typhoid  bacillus  

1  :  2100 

1  :300 

Cholera  spirillum  

1  :  5500 

1  •  1350 

Chromic  Acid. — In  Koch's  experiments  a  one-per-cent  solution 
destroyed  anthrax  spores  in  from  one  to  two  days.  In  the  propor- 
tion of  1  :  5,000  it  prevents  the  development  of  putrefactive  bacteria 
(Miquel). 

Osmic  Acid. — A  solution  of  one  per  cent  kills  anthrax  spores  in 
twenty-four  hours  (Koch).  It  is  an  antiseptic  in  the  proportion  of 
1  : 6,666  (Miquel). 

Phosphoric  Acid. — Exposure  for  four  or  five  hours  to  a  solution 


182  ACTION   OF    ACIDS   AND    ALKALIES. 

containing  0.3  per  cent  destroys  the  typhoid  bacillus,  and  0.183  per 
cent  the  cholera  spirillum  (Kitasato).  The  acid  used  contained  0.152 
gramme  H3PO4  in  one  cubic  centimetre. 

Acetic  Acid.— A  five-per-cent  solution  failed  to  kill  anthrax 
spores  after  five  days'  exposure  (Koch).  In  Abbott's  experiments 
glacial  acetic  acid  in  fifty-per-cent  solution  failed  in  two  hours  to  kill 
anthrax  spores,  but  micrococci  were  killed  by  two  hours7  exposure  to 
a  one-per-cent  solution.  A  solution  of  1  : 300  of  glacial  acetic  acid 
destroys  the  cholera  spirillum  in  half  an  hour  (Van  Ermengem).  In 
the  proportion  of  0. 25  per  cent  it  restrains  the  growth  of  the  typhoid 
bacillus,  and  0. 3  per  cent  destroys  its  vitality  after  five  hours'  expo- 
sure ;  the  cholera  spirillum  fails  to  grow  in  presence  of  0.132  per  cent 
and.  is  destroyed  by  0.2  per  cent  (Kitasato). 

Lactic  Acid. — The  bacillus  of  typhoid  fever  is  killed  in  five  hours 
by  a  solution  containing  0. 4  per  cent,  the  cholera  spirillum  by  0. 3  per 
cent  (Kitasato). 

Citric  Acid. — The  bacillus  of  typhoid  fever  is  killed  in  five  hours 
by  0.43  per  cent,  the  cholera  spirillum  by  0.3  per  cent  (Kitasato). 
The  cholera  spirillum  is  killed  in  half  an  hour  by  1  : 200  (Van  Er- 
mengem). 

Oxalic  Acid. — The  typhoid  bacillus  requires  a  solution  of  0.36 
per  cent,  the  cholera  spirillum  one  of  0. 28  per  cent,  to  destroy  vitality 
in  five  hours  (Kitasato). 

Boracic  Acid. — In  the  writer's  experiments  (1883)  a  saturated 
solution  failed  to  kill  pus  cocci  in  two  hours.  A  five-per-cent  solu- 
tion failed  to  destroy  anthrax  spores  in  five  days  (Koch).  The 
typhoid  bacillus  is  killed  in  five  hours  by  2.7  percent,  the  cholera 
spirillum  by  1.5  per  cent  (Kitasato).  According  to  Arloing,  Corne- 
vin,  and  Thomas,  the  fresh  virus  of  symptomatic  anthrax  requires 
exposure  to  a  twenty-per-cent  solution  for  forty-eight  hours  for  the 
destruction  of  vitality.  Boracic  acid  acts  as  an  antiseptic  in  the  pro- 
portion of  1  : 143  (Miquel). 

Salicylic  Acid. — In  the  writer's  experiments  this  agent  was  dis- 
solved by  the  addition  of  sodium  biborate,  which  by  itself  has  no 
germicidal  power.  A  two-per-cent  solution  was  found  to  destroy  pus 
cocci  in  two  hours.  Dissolved  in  oil  or  in  alcohol  a  five-per-cent  so- 
lution does  not  destroy  anthrax  spores  (Koch).  Micrococci  are  de- 
stroyed by  solutions  containing  1  : 400  (Abbott).  The  typhoid  bacillus 
is  killed  in  five  hours  by  1.6  per  cent,  the  cholera  spirillum  by  1.3  per 
cent  (Kitasato).  A  one-per-cent  solution  destroys  Micrococcus  Pas- 
teuri  in  half  an  hour  (Sternberg).  It  is  an  antiseptic  in  the  propor- 
tion of  1  : 1,000  (Miquel).  A  solution  of  2.5  percent  kills  the  tubercle 
bacillus  in  six  hours  (Yersin).  In  the  proportion  of  1  : 300  it  destroys 
the  cholera  spirillum  in  half  an  hour  (Van  Ermengem). 


ACTION   OF   ACIDS   AXD    ALKALIES. 

Benzole  Acid. — According  to  Miquel,  this  acid  restrains  the  de- 
velopment of  putrefactive  bacteria  when  present  in  bouillon  in  the 
proportion  of  1: 909.  In  the  proportion  of  1  :  2,000  it  retards  the  de- 
velopment of  anthrax  spores  (Koch). 

Formic  Acid. — The  typhoid  bacillus  is  restrained  in  its  growth  by 
0.25  per  cent,  and  is  killed  in  five  hours  by  0.35  per  cent,  the  cholera 
spirillum  by  0.22  per  cent  (Kitasato). 

Tannic  Acid. — A  solution  of  one  per  cent  kills  Micrococcus  Pas- 
teuri  in  the  blood  of  a  rabbit  in  half  an  hour  (Sternberg).  A  five- 
per-cent  solution  failed  in  ten  days  to  destroy  anthrax  spores  (Koch). 
A  twenty-per-cent  solution  failed  in  two  hours  to  destroy  the  vitality 
of  spores  of  the  anthrax  bacillus  or  of  Bacillus  subtilis  (Abbott). 
Micrococci  are  destroyed  by  1  : 400,  and  1  : 800  failed  (Abbott).  A 
twenty-per-cent  solution  has  no  effect  upon  the  virus  of  symptomatic 
anthrax  (Arloing,  Cornevin,  and  Thomas).  A  solution  of  1.66  per 
cent  kills  the  typhoid  bacillus  in  five  hours,  and  1.5  per  cent  the 
cholera  bacillus  in  the  same  time  (Kitasato).  It  restrains  the  devel- 
opment of  putrefactive  bacteria  in  the  proportion  of  1  :  207  (Miquel). 

Tartaric  Acid. — A  twenty-per-cent  solution  of  this  acid  fails, 
after  two  hours'  exposure,  to  destroy  the  spores  of  Bacillus  anthracis 
or  Bacillus  subtilis.  Micrococci  are  killed  by  two  hours'  exposure  in 
a  solution  containing  1  :  400  (Abbott). 

Malic  Acid. — This  was  found  by  Kitasato  to  correspond  with 
citric  acid  in  its  germicidal  power. 

Valerianic  Acid. — A  five-per-cent  solution  in  ether  failed  in  five 
days  to  destroy  anthrax  spores  (Koch). 

Oleic  Acid. — A  solution  of  five  percent  in  ether  does  not  destroy 
anthrax  spores  in  five  days  (Koch). 

Thymic  Acid. — In  the  proportion  of  1  :  500  this  acid  prevents  the 
putrefactive  decomposition  of  beef  tea  (Miquel). 

Butyric  Acid. — Five  days'  immersion  in  this  acid  failed  to  de- 
stroy anthrax  spores  (Koch). 

Arsenious  Acid. — A  one-per-cent  solution  destroys  the  vitality 
of  anthrax  spores  in  ten  days,  but  failed  to  do  so  in  six  days  (Koch). 
In  the  proportion  of  1  : 166  it  prevents  putrefactive  changes  in  bouillon 
(Miquel). 

Gallic  Acid. — Abbott  found  this  acid  to  destroy  the  bacteria  in 
broken-down  beef  tea  in  the  proportion  of  2.37  per  cent,  but  it  failed 
to  destroy  anthrax  spores  in  two  hours  in  the  same  proportion.  Mi- 
crococci were  killed  in  two  hours  by  1  : 142,  while  1  :  250  failed. 

ALKALIES. 

Potassium  Hydroxide,  KHO. — In  the  writer's  experiments  aten- 
per-cent  solution  of  caustic  potash  was  fatal  to  pus  cocci,  and  an 


184  ACTION    OF   ACIDS   AND   ALKALIES. 

eight-per-cent  solution  failed — two  hours'  exposure.  Exposure  for 
twenty-four  hours  to  a  ten-per-cent  solution  failed  to  kill  the  tubercle 
bacillus  (Schill  and  Fischer).  A  solution  of  one  per  cent  kills  the 
anthrax  bacillus,  the  bacillus  of  rothlauf,  and  several  others  (Jager). 
The  addition  of  0. 14  per  cent  restrains  the  development  of  the  typhoid 
bacillus,  and  0.18  per  cent  kills  this  bacillus  in  four  or  five  hours;  the 
cholera  spirillum  failed  to  grow  in  cultures  containing  0.18  per  cent 
and  was  killed  by  0.237  per  cent  in  the  same  time  (Kitasato). 

Sodium  Hydroxide,  NaHO. — The  experiments  of  Jager  and  of 
Kitasato  show  that  soda  has  about  the  same  germicidal  power  as 
caustic  potash.  Boer  obtained  the  following  results  with  bouillon 
cultures  after  two  hours'  exposure :  Anthrax  bacillus,  1  :  450  ;  diph- 
theria bacillus,  1  : 300  ;  glanders  bacillus,  1  : 150 ;  typhoid  bacillus, 
1  : 190  ;  cholera  spirillum,  1  : 150.  In  about  one-half  the  amount 
required  to  destroy  vitality  the  development  of  the  above-named  bac- 
teria was  prevented.  In  the  proportion  of  1  :  56  it  acts  as  an  anti- 
septic (Miquel). 

Ammonia,  NH3. — In  Kitasato's  experiments  the  typhoid  bacillus 
was  destroyed  in  five  hours  by  0.3  per  cent  of  NH3,  and  the  cholera 
spirillum  by  about  the  same  amount.  Boer  obtained  the  following 
results,  the  time  of  exposure  being  two  hours  :  Anthrax  bacillus, 
1: 300  ;  diphtheria  bacillus,  1  :  250  ;  glanders  bacillus,  1  : 250  ;  typhoid 
bacillus,  1 : 200 ;  cholera  spirillum,  1  : 350.  The  growth  of  the  an- 
thrax bacillus  and  of  the  diphtheria  bacillus  in  culture  solutions  was 
prevented  by  1  :  650. 

Calcium  Hydroxide,  Ca2HO. — According  to  Kitasato,  the  ty- 
phoid bacillus  and  the  cholera  spirillum,  in  bouillon  cultures,  are 
killed  in  four  or  five  hours  by  the  addition  of  0. 1  per  cent  of  calcium 
oxide.  Liborius  had  previously  reported  still  more  favorable  results, 
but  his  bouillon  cultures  were  largely  diluted  with  distilled  water. 
From  a  practical  point  of  view  the  experiments  of  Pfuhl  are  more 
valuable.  Calcium  hydrate  was  added  to  the  dejections  of  typhoid 
patients.  When  added  in  the  proportion  of  three  per  cent  steriliza- 
tion was  effected  in  six  hours,  and  by  six  per  cent  in  two  hours. 
When  milk  of  lime  containing  twenty  per  cent  of  calcium  hydrate 
was  used  the  results  were  still  more  favorable,  the  typhoid  bacillus 
and  cholera  spirillum  being  killed  in  one  hour  by  the  addition  of 
two  per  cent  of  the  disinfectant.  The  practical  value  of  lime-wash 
applied  to  walls  has  been  determined  by  Jager.  Silk  threads  soaked 
in  cultures  of  various  pathogenic  bacteria  were  attached  to  boards 
and  the  lime- wash  applied  with  a  cameFs-hair  brush.  Anthrax  ba- 
cilli (without  spores),  the  glanders  bacillus,  Staphylococcus  pyogene? 
aureus,  and  several  other  pathogenic  bacteria  were  killed  by  a  single 
application  after  twenty-four  hours,  but  the  tubercle  bacillus  was  not 


ACTION   OF   ACIDS   AND    ALKALIES.  185 

killed  by  three  successive  applications.  In  the  writer's  experiments 
(1885)  the  typhoid  bacillus  and  Staphylococcus  pyogenes  aureus  were 
killed  in  two  hours  by  a  solution  containing  1 : 40  of  calcium  oxide, 
and  1 :  80  failed.  Spores  of  the  anthrax  bacillus  and  of  several  other 
spore-forming  species  were  not  killed  by  two  hours'  exposure  to  a 
milk  of  lime  containing  twenty  per  cent  of  calcium  oxide. 

Potash  Soap  has  been  shown  by  Jolles  (1895)  to  have  considerable 
germicidal  value.  In  experiments  with  a  soap  containing  67.44 
per  cent  of  fat  acids,  10.4  per  cent  of  combined  alkali,  and  0.041 
per  cent  of  free  alkali,  the  following  results  were  obtained :  The 
typhoid  bacillus  was  destroyed  at  18°  C.  by  a  one-per-cent  solution 
in  twenty -four  hours;  by  a  six-per-cent  solution  in  thirty  minutes. 
The  Bacillus  coli  communis  required  somewhat  stronger  solutions  or 
longer  exposure — eight-per-cent  solution  required  thirty  minutes. 
These  experiments  show  that  scrubbing  with  soap  and  water  is  a 
reliable  method  of  disinfecting  surfaces.  Solutions  of  potash — com- 
mon lye — or  of  soda  also  are  useful  for  certain  purposes  in  domes- 
tic disinfection,  and  scientific  researches  justify  the  continued  use  of 
the  cleansing  methods  which  have  heretofore  been  in  use  by  careful 
housewives. 


X. 
ACTION  OF  SALTS. 

WHILE  some  of  the  metallic  salts,  and  especially  those  of  mer- 
cury, silver,  and  gold,  have  remarkable  germicidal  power,  others, 
even  in  concentrated  solutions,  do  not  destroy  the  vitality  of  bacteria 
exposed  to  their  action.  For  convenience  of  reference  we  shall  con- 
sider the  agents  in  this  group  in  alphabetical  order,  but  first  we  give 
Miquel's  tables  of  antiseptic  value.  This  author  recognizes  the  im- 
portance of  experiments  to  determine  the  restraining  power  of  chem- 
ical agents  for  various  species  of  pathogenic  bacteria,  but  says  :  "  As 
to  me,  faithful  to  a  plan  I  adopted  at  the  outset,  I  will  treat  the  sub- 
ject in  a  more  general  manner  by  making  known  simply  the  mini- 
mum weight  of  the  substances  capable  of  preventing  the  evolution  of 
any  bacteria  or  germs.  The  method  adopted  is  very  simple.  To  a 
liquid  always  comparable  to  itself  it  is  sufficient  at  first  to  add  a 
known  weight  of  the  antiseptic  and  some  atmospheric  germs  or  adult 
bacteria,  and  to  vary  the  quantity  of  the  antiseptic  until  the  amount 
is  ascertained  which  will  preserve  indefinitely  the  liquid  from  putre- 
faction. In  order  to  obtain  germs  of  all  kinds  in  a  dry  state  it  suf- 
fices to  take  them,  where  they  are  most  abundant,  in  the  dust  col- 
lected in  the  interior  of  houses  or  of  hospitals;  and  to  procure  a 
variety  of  adult  bacteria  we  may  take  the  water  of  sewers. " 

SUBSTANCES   EMINENTLY  ANTISEPTIC. 

Efficient  in  the 
proportion  of— 

Mercuric  iodide,          .  .  .  .  .  1 : 40000 

Silver  iodide,         ......  1:33000 

Hydrogen  peroxide,  .  .  .  .  .  1 : 20000 

Mercuric  chloride,  .  .  .  .  .  1 : 14300 

Silver  nitrate,  .  .  .  .  .  1 : 12500 

SUBSTANCES   VERY   STRONGLY   ANTISEPTIC. 

Osmicacid,            ......  1:6666 

Chromic  acid,              .             .             .            .            .  .        1 : 5000 

Chlorine,                ......  1:4000 

Iodine,  .......        1:4000 

Chloride  of  gold,  .             .....  1:4000 

Bichloride  of  platinum,         .            .            .            .  .        1 : 3333 

Hydrocyanic  acid,            .            .            .            .             .  1 : 2500 


ACTION   OF    SALTS.  187 

Bromine,          .......  1:1666 

Cupric  chloride,    .  .  .  .  .  .  1 : 1428 

Thymol,  .......  1:1340 

Cupric  sulphate,   .  .  .  .  .  .  1 : 1111 

Salicylic  acid,  .  .  .  .  .  1 : 1000 


SUBSTANCES   STRONGLY  ANTISEPTIC. 

Benzoic  acid,         .            .  .            .            .             .             1 : 909 

Potassium  bichromate,  .             .             .             .                    1 : 909 

Potassium  cyanide,           .  .             .             .             .             1 : 909 

Aluminum  chloride,  .....       1:714 

Ammonia,              .             .  .             .             .             .             1 : 714 

Zinc  chloride,               .  .             .             .             .             .1:526 

Mineral  acids,        .  ...          1:500  to  1:333 

Thymicacid,  .             .  .            .             .            .             .1:500 

Lead  chloride,       .            .  .            .            .            .             1 : 500 

Nitrate  of  cobalt,         .  .            .            .            .            .1:476 

Sulphate  of  nickel,            .  .            .            .            .             1:400 

Nitrate  of  uranium,    .  .             .             .             .             .       1 : 356 

Carbolic  acid,        .            .  .            .            .            .             1:333 

Potassium  permanganate,  .             .             .             .                    1 : 285 

Lead  nitrate,          .             .  .            .            .            .             1 : 277 

Alum,.             .             .  .             .             .             .             .1:222 

Tannin, 1:207 


SUBSTANCES   MODERATELY   ANTISEPTIC. 

Bromhydrate  of  quinine,       .....  1:182 

Arsenious  acid,     .  .  .  .  .  .  1 : 166 

Boracic  acid,   .  .  .  .  .  .  1 : 143 

Sulphate  of  strychnia,     .  .  .  .  .  1 : 143 

Arsenite  of  soda,         ......  1:111 

Hydrate  of  chloral,  .  .  .  .    „  1:107 

Salicylate  of  soda,      .  .  .  .  .  .  1 : 100 

Ferrous  sulphate,  .  .  .  .  .  1 : 90 

Caustic  soda,   .  .  .  .  .  .  1 : 56 


SUBSTANCES  FREELY  ANTISEPTIC. 

Perchloride  of  manganese,  .             .             .             .             1 : 40 

Calcium  chloride,       .  .            .            .            .             .       1 : 25 

Sodium  borate,      .             .  .            .             .             .             1 : 14 

Muriate  of  morphia,"  .  .             .             .             .                    1 : 13 

Strontium  chloride,          .  .             .             .             .              1 : 12 

Lithium  chloride,        .  .             .             .             .                    -1 : 11 

Barium  chloride,  .             .  .             .             .             .              1 : 10 

Alcohol,            .  1:10 


SUBSTANCES   VERY   FEEBLY  ANTISEPTIC. 

Ammonium  chloride,  .             .             .             .             .             1:9 

Potassium  arsenite,    .  .             .             .             .             .1:8 

Potassium  iodide,  .            .             .            .             .             1:7 

Sodium  chloride,         .  .            .            .            .             .1:6 

Glycerin  (sp.  gr.  1.25),  .             .             .             .             .              1:4 

Ammonium  sulphate,  .             .             .             .             .1:4 

Sodium  hyposulphite,  .            .            .            .            .             1:3 


188  ACTION    OF   SALTS. 

ANTISEPTIC    AND    GERMICIDAL    VALUE   OF   VARIOUS    SALTS, 
ARRANGED   ALPHABETICALLY. 

Alum. — : Antiseptic  in  the  proportion  of  1  :  222  (Miquel). 

Aluminum  Acetate. — According  to  De  la  Croix,  this  salt  is  an 
antiseptic  in  the  proportion  of  1  :  6,310.  Kuhn  found  it  to  be  anti- 
septic in  1  : 5,250. 

Aluminum  Chloride. — Antiseptic  in  the  proportion  of  1  :  714 
(Miquel). 

Ammonium  Carbonate. — When  present  in  the  proportion  of 
1  : 125  it  restrains  the  development  of  typhoid  bacilli,  and  in  five 
hours'  time  it  kills  these  bacilli  in  the  proportion  of  1  : 100 ;  the 
cholera  spirillum  is  killed  in  the  same  time  by  1  :  77  (Kitasato). 

Ammonium  Chloride. — Antiseptic  in  the  proportion  of  1:0 
(Miquel).  A  five-per-cent  solution  does  not  kill  anthrax  spores  in 
twenty-five  days  (Koch). 

Ammonium  Fluosilicate. — The  bacillus  of  anthrax  and  of  ty- 
phoid fever  fail  to  grow  in  nutrient  gelatin  containing  1  : 1,000,  and 
a  two-per-cent  solution  kills  anthrax  spores  in  one-quarter  to  three- 
quarters  of  an  hour  (Faktor). 

Ammonium  Sulphate. — Antiseptic  in  the  proportion  of  1:4 
(Miquel).  A  five-per-cent  solution  failed  in  two  days  to  kill  an- 
thrax spores,  but  was  effective  in  five  days  (Koch). 

Barium  Chloride  is  an  antiseptic  in  the  proportion  of  1  : 10 
(Miquel). 

Calcium  Chloride  is  an  antiseptic  in  the  proportion  of  1  : 25 
(Miquel).  A  saturated  solution  does  not  destroy  anthrax  spores 
(Koch). 

Calcium  Hypochlorite. — This  is  a  powerful  germicidal  agent 
and  has  great  value  as  a  practical  disinfectant.  Good  chloride  of 
lime  contains  from  twenty-five  to  thirty  per  cent  of  available  chlo- 
rine as  hypochlorite.  The  experiments  made  by  the  Committee  on 
Disinfectants  of  the  American  Public  Health  Association  in  1885 
showed  that  a  solution  containing  0.25  per  cent  of  chlorine  as  hypo- 
chlorite is  an  effective  germicide,  even  when  allowed  to  act  only 
for  one  or  two  minutes.  In  Bolton's  experiments  a  solution  of  chlo- 
ride of  lime  of  1  :2,000  (available  chlorine  0.015)  destroyed  the  ty- 
phoid bacillus  and  the  cholera  spirillum  in  two  hours.  For  the  de- 
struction of  anthrax  spores  a  one-per-cent  solution  was  required 
(available  chlorine  0.3  per  cent).  Nissen  found  that  the  typhoid 
bacillus  and  the  cholera  spirillum  are  destroyed  with  certainty  in 
five  minutes  by  a  solution  containing  0.12  percent,  anthrax  bacilli 
in  one  minute  by  0. 1  per  cent,  Staphylococcus  pyogenes  aureus  in 
one  minute  by  0.2  per  cent,  anthrax  spores  in  thirty  minutes  by  a 


ACTION   OF   SALTS.  180 

five-per-cent  solution  and  in  seventy  minutes  by  a  one-per-cent  solu- 
tion. Experiments  made  by  the  same  author  upon  the  sterilization 
of  f  aeces  showed  that  0. 5  per  cent  to  one  per  cent  could  be  relied  upon 
to  destroy  the  typhoid  bacillus  or  the  cholera  spirillum  in  faeces  in 
ten  minutes. 

Chloral  Hydrate. — Antiseptic  in  the  proportion  of  1  : 107  (Mi- 
quel).  A  twenty-per*cent  solution  destroys  pus  cocci  in  two  hours 
(Sternberg). 

Cupric  Chloride. — Antiseptic  in  the  proportion  of  1  •  1,428 
(Miquel). 

Cupric  Sulphate. — Antiseptic  in  the  proportion  of  1  :  111  (Mi- 
quel). Kills  the  cholera  spirillum  in  the  proportion  of  1  :  3,000  in 
ten  minutes  (Nicati  and  Rietsch).  Destroys  the  cholera  spirillum  in 
bouillon  cultures  in  less  than  half  an  hour  in  1  :  600,  and  in  four 
hours  in  1  : 1,000  ;  cultures  in  blood  serum  require  1  :  200  (Van  Er- 
mengem).  A  solution  of  1  :  20  kills  the  typhoid  bacillus  in  ten  min- 
utes (Leitz).  This  salt  failed,  in  the  writer's  experiments,  to  kill  the 
spores  of  Bacillus  anthracis  and  Bacillus  subtilis  in  two  hours'  time 
in  a  twenty-per-cent  solution.  In  Koch's  experiments  a  five-per-cent 
solution  failed  to  kill  anthrax  spores  in  ten  days.  Kills  pus  micro- 
cocci  in  two  hours  in  the  proportion  of  1  :  200  (Sternberg).  In  Bol- 
ton's  experiments  made  for  the  Gommittee  on  Disinfectants  of  the 
American  Public  Health  Association  the  following  results  were  ob- 
tained: Recent  cultures  in  bouillon,  time  of  exposure  two  hours  :  Ba- 
cillus of  typhoid  fever,  1  :  200;  cholera  spirillum,  1  :  500;  Bacillus  pyo- 
cyanus,  1 :200;  Brieger's  bacillus,  1  :200;  Emmerich's  bacillus,  1  :  200; 
Staphylococcus  pyogenes  aureus,  1  : 100  ;  Staphylococcus  pyogenes 
citreus,  1  : 100;  Staphylococcus  pyogenes  albus,  1  :  200;  Streptococcus 
pyogenes,  1  : 500.  When  ten  per  cent  of  dried  egg  albumin  was 
added  to  a  recent  culture  in  bouillon  of  the  typhoid  bacillus  the 
amount  required  to  insure  sterilization  was  1  : 10. 

In  the  report  of  the  Committee  on  Disinfectants  of  the  American 
Public  Health  Association  this  agent  is  recommended  in  "  a  solu- 
tion of  two  to  five  per  cent  for  the  destruction  of  infectious  material 
not  containing  spores."  The  experimental  data  above  given  show 
that  this  is  a  liberal  allowance  for  material  which  does  not  contain 
an  excessive  amount  of  albumin.  In  the  experiments  of  Leitz  the 
typhoid  bacillus  in  cultures  was  destroyed  in  ten  minutes  by  a  five- 
per-cent  solution. 

Ferric  Chloride. — A  five-per-cent  solution  failed  in  two  days  to 
destroy  anthrax  spores,  but  was  effective  in  five  days  (Koch).  • 

Ferrous  Sulphate. — In  the  writer's  experiments  (1883)  a  solution 
of  twenty  per  cent  failed  to  destroy  micrococci  and  putrefactive  bac- 
teria. In  a  more  recent  experiment  ten  per  cent  failed  to  kill  pus 


1'JU  ACTION   OF   SALTS. 

cocci,  but  was  fatal  to  Micrococcus  tetragenus — two  hours'  exposure. 
Koch  found  that  a  five-per-cent  solution  failed  to  destroy  anthrax 
spores  in  six  days.  Exposure  to  a  twenty-per-cent  solution  for  forty- 
eight  hours  does  not  destroy  the  virus  of  symptomatic  anthrax  (Ar- 
loing,  Cornevin,  and  Thomas).  In  the  experiments  of  Jager  immer- 
sion in  a  solution  of  1  :  3  destroyed  the  infective  virulence  of  certain 
pathogenic  bacteria  (fowl  cholera,  rothlauf,  glanders),  as  tested  by 
injection  into  mice,  but  failed  to  kill  anthrax  spores  and  tubercle  ba- 
cilli. The  antiseptic  power  of  ferrous  sulphate  is  placed  by  Miquel 
at  1  :  90.  In  the  writer's  experiments  1  :  200  prevented  the  develop- 
ment of  micrococci  and  of  putrefactive  bacteria  in  bouillon  placed 
in  the  incubating  oven  for  forty-eight  hours.  Leitz  found  that  a 
five-per-cent  solution  required  three  days'  exposure  for  the  destruc- 
tion of  the  typhoid  bacillus. 

Gold  Chloride. — Antiseptic  in  the  proportion  of  1  :  4,000  (Miquel). 
Boer  has  made  extended  experiments  with  the  chloride  of  gold  and 
sodium.  We  give  his  results  below.  In  his  disinfection  experi- 
ments a  bouillon  culture  which  had  been  in  the  incubating  oven  for 
twenty-four  hours  was  used,  and  the  time  of  exposure  was  two  hours. 


Restrains 
development. 

Destroys 
vitality. 

Anthrax  bacillus   .  .  ,  

1  :  40000 

1  :  8000 

Diphtheria  bacillus  

1  :  40000 

1  :  1  000 

Glanders  bacillus     

1  :  1  5000 

1  •  400 

Typhoid  bacillus  .   

1  :  20000 

1  :500 

Cholera  spirillum  

1  :  25000 

1  :  1000 

Lead  Chloride. — Antiseptic  in  the  proportion  of  1  :  500  (Miquel). 

Lead  Nitrate. — Antiseptic  in  the  proportion  of  1  :  277  (Miquel). 

Lithium  Chloride. — Antiseptic  in  the  proportion  of  1  : 11  (Mi- 
quel). 

Manganese  Protochloride. — Antiseptic  in  the  proportion  of  1:40 
(Miquel). 

Mercuric  Chloride. — Koch's  experiments  (1881)  gave  the  follow- 
ing results  :  A  solution  of  1  : 1,000  destroys  anthrax  spores  in  a  few 
minutes,  and  1  : 10,000  is  effective  after  a  more  prolonged  exposure. 
The  writer  (1884)  obtained  similar  results — 1  : 10,000  destroyed  the 
spores  of  Bacillus  anthracis  and  of  Bacillus  subtilis  in  two  hours. 
More  recent  experiments  indicate  that  failure  to  grow  in  culture  so- 
lutions cannot  be  accepted  as  evidence  of  the  destruction  of  vitality 
in  the  case  of  spores  exposed  to  the  action  of  this  agent,  unless  due 
precautions  are  taken  to  exclude  the  restraining  influence  of  the  small 
amount  of  mercuric  chloride  which  remains  attached  to  the  spores. 
Koch  had  ascertained  that  the  development  of  spores  is  restrained  by 


ACTION   OF   SALTS.  1U1 

the  presence  of  1  : 300,000  in  a  culture  medium,  and  Geppert  has  re- 
cently shown  that  even  so  small  an  amount  as  1  :  2,000,000  will  pre- 
vent the  development  of  spores  the  vitality  of  which  has  been  reduced 
by  the  action  of  a  strong  solution  (1  : 1,000).  When  this  restraining 
action  is  entirely  neutralized  by  washing  the  spores  in  a  solution  con- 
taining ammonium  sulphide  it  requires,  according  to  Geppert,  a  solu- 
tion of  1:1,000  acting  for  one  hour  to  completely  destroy  the  vitality 
of  anthrax  spores.  Frankel  found  that  a  solution  of  1  : 1,000  was 
effective  in  half  an  hour.  The  typhoid  bacillus,  the  bacillus  of  mouse 
septica3mia,  and  the  cholera  spirillum,  in  bouillon  cultures  and  in 
cultures  in  flesh-peptone-gelatin,  are  destroyed  in  two  hours  by 
1  : 10,000  ;  but  in  a  bouillon  culture  to  which  ten  per  cent  of  dried 
egg  albumin  was  added  a  one-per-cent  solution  was  required  to  de- 
stroy the  typhoid  bacillus  in  the  same  time  (Bolton).  According  to 
Van  Ermengem,  cultures  of  the  cholera  spirillum  in  bouillon  are  steril- 
ized in  half  an  hour  by  1  :  60,000,  but  cultures  in  blood  serum  require 
1  :  800  to  1  : 1,000.  In  experiments  upon  tuberculous  sputum  Schill 
and  Fischer  found  that  exposure  of  fresh  sputum  to  an  equal  amount 
of  a  1  : 2,000  solution  for  twenty-four  hours  failed  to  disinfect  it,  as 
shown  by  inoculation  experiments  in  guinea-pigs.  The  antiseptic 
power  of  mercuric  chloride  is  given  by  Miquel  as  1  : 14,300.  In  the 
writer's  experiments  1  : 33,000  was  found  to  prevent  the  development 
of  putrefactive  bacteria  in  bouillon,  but  a  minute  bacillus  contained  in 
broken-down  beef  infusion  multiplied,  after  several  days,  in  1  :  20,000. 
The  pus  cocci  were  restrained  in  their  development  by  1  :  30,000. 

In  Behring's  experiments  the  anthrax  bacillus  and  cholera  spiril- 
lum were  killed  in  one  hour  by  1  : 100,000  when  the  temperature 
was  36°  C.,  but  at  a  temperature  of  3°  C.  the  proportion  required 
was  1  : 25,000.  The  same  author  states  that  at  22°  C.  Staphylo- 
coccus  aureus  in  bouillon  is  not  always  killed  in  twenty-five  minutes 
by  1  : 1,000. 

Abbott  (1891)  has  shown  that  a  1 : 1,000  solution  does  not  always 
destroy  Staphylococcus  pyogenes  aureus  in  five  minutes.  He  says : 
"  Frequently  all  the  organisms  would  be  destroyed  after  five  minutes' 
exposure,  but  almost  as  often  a  certain  few  would  resist  for  that 
length  of  time,  and  even  longer,  going  in  some  cases  to  ten,  twenty, 
and  even  thirty  minutes." 

According  to  Yersin,  a  solution  of  1  : 1,000  kills  the  tubercle  bacil- 
lus in  one  minute. 

We  might  add  considerably  to  the  experimental  data  given,  but 
the  results  already  recorded  are  sufficient  to  show  the  value  of  this 
agent  as  an  antiseptic  and  germicide,  and  justify  its  use  for  general 
purposes  of  disinfection  in  the  proportion  of  1  : 500  or  1  :  lrOOO  for 
material  containing  spores,  and  in  the  proportion  of  1  : 2,000  to 


192  ACTION   OF   SALTS. 

1  :  5,000  for  pathogenic  bacteria  in  the  absence  of  spores;  due  regard 
being  had  to  the  fact  that  the  presence  of  albumin  very  materially 
reduces  its  germicidal  potency,  and  that  it  may  be  decomposed  and 
neutralized  by  alkalies  and  their  carbonates,  by  hydrosulphuric  acid, 
and  by  many  other  substances. 

The  albuminate  of  mercury,  as  has  been  shown  by  Lister,  is  solu- 
ble in  an  excess  of  albumin,  and,  according  to  Behring,  is  just  as 
effective  as  an  aqueous  solution  containing  the  same  amount  of  sub- 
limate when  dissolved  in  an  albuminous  liquid  like  blood  serum  (?). 

In  practice  the  addition  of  a  mineral  acid  to  sublimate  solutions, 
or  of  sodium,  potassium,  or  ammonium  chloride,  is  to  be  recom- 
mended, to  prevent  the  precipitation  of  the  mercuric  chloride  by  al- 
bumin in  fluids  containing  it.  Behring  recommends  the  addition 
of  five  parts  of  sodium  or  potassium  chloride  to  one  of  the  subli- 
mate. Such  a  solution  is  more  stable  than  a  simple  solution  of  sub- 
limate, and  no  precipitate  is  formed  by  the  addition  of  alkalies  or  by 
albumin. 

The  same  result  is  obtained,  according  to  La  Place,  by  the  addi- 
tion of  five  parts  of  hydrochloric  or  tartaric  acid  to  one  part  of  sub- 
limate in  aqueous  solution. 

Mercuric  Cyanide,  Hg(CN)2,  and  the  Oxy cyanide  of  mercury 
have  been  tested,  with  the  following  results  :  Staphylococcus  aureus 
is  destroyed  in  five  minutes  by  1  : 100,  in  one  hour  by  1  : 1,000,  in 
two  hours  by  1  : 1,500  (Chibret).  The  development  of  Bacillus  an- 
thracis  in  culture  solutions  is  prevented  by  the  presence  of  cyanide 
of  mercury  in  the  proportion  of  1  :  25,000,  and  by  the  oxy cyanide  by 
1  : 16,000  (Behring). 

Boer  obtained  the  following  results  with  the  oxycyanide — cul- 
tures in  bouillon,  twenty-four  hours  in  incubating  oven,  time  of 
exposure  two  hours  : 


Restrained 
development. 

Destroyed 
vitality. 

Anthrax  bacillus  

1  :  80000 

1  :  40000 

Diphtheria  bacillus  

1  :  80000 

1  •  40000 

Glanders  bacillus  

1  :  60000 

1  :  30000 

Typhoid  bacillus  •  ,  

1  :  60000 

1  :  30000 

Cholera  spirillum  

1  :  90000 

1  :  60000 

Mercuric  Iodide. — The  antiseptic  value  of  this  salt  is  placed  by 
Miquel  at  1  : 40,000,  which  is  more  than  double  that  given  by  the 
same  author  to  the  bichloride.  In  the  writer's  experiments  upon  the 
antiseptic  value  of  salts  and  oxides  of  mercury  the  following  results 
were  obtained  : 


ACTIOX   OF    SALTS.  193 


Active. 

Failed. 

Biniodide  of  mercury  

1  :  20000 

1  :  40000 

Bichloride    

1  :  15000 

1  :  20000 

Protiodide  

1  .  10000 

1  :  20000 

Yellow  oxide  

1  :  1000 

1  :  2000 

Black  oxide  

1  :500 

1    1000 

Morphia  Hydrochlorate. — Antiseptic  in  the  proportion  of  1  : 13 
(Miquel). 

Nickel  Sulphate. — Antiseptic  in  the  proportion  of  1  :  400  (Mi- 
quel). 

Platinum  Bichloride. — Antiseptic  in  the  proportion  of  1  :  3,333 
(Miquel). 

Potassium  Acetate. — A  saturated  solution  of  this  salt  failed  to 
kill  anthrax  spores  in  ten  days  (Koch). 

Potassium  Arsenite. — In  the  writer's  experiments  Fowler's  solu- 
tion failed  to  kill  micrococci  in  two  hours  in  the  proportion  of  four 
per  cent.  Miquel  places  the  antiseptic  value  of  potassium  arsenite 
at  1  : 8. 

Potassium  Bichromate. — A  five-per-cent  solution  failed  in  two 
days  to  destroy  anthrax  spores  (Koch).  Efficient  as  an  antiseptic  in 
the  proportion  of  1  :  909  (Miquel). 

Potassium  Bromide. — The  bacillus  of  typhoid  fever  and  the 
cholera  spirillum  fail  to  grow  in  culture  solutions  containing  9  to 
10.6  per  cent,  and  are  killed  in  four  or  five  hours  by  ten  to  twelve 
per  cent  (Kitasato). 

Potassium  Carbonate. — The  development  of  the  typhoid  bacil- 
lus and  of  the  cholera  spirillum  is  prevented  by  0.74  to  0.81  per 
cent,  and  these  bacteria  are  killed  in  five  hours  by  1  per  cent  (Kita- 
sato). 

Potassium  Chlorate. — In  the  writer's  experiments  a  four-per- 
cent solution  failed  in  two  hours  to  kill  Micrococcus  Pasteuri.  A 
five-per-cent  solution  failed  in  six  days  to  destroy  anthrax  spores 
(Koch). 

Potassium  Chromate. — A  five-per-cent  solution  failed  to  kill 
anthrax  spores  in  five  days  (Koch). 

Potassium  Cyanide. — Antiseptic  in  the  proportion  of  1  :  909 
(Miquel). 

Potassium  Iodide. — A  solution  of  five  per  cent  does  not  destroy 
anthrax  spores  in  eighty  days.  (Koch).  Putrefactive  bacteria  in 
broken-down  beef  infusion  are  not  destroyed  by  two  hours'  exposure 
in  a  twenty-per-cent  solution  (Sternberg).  The  typhoid  bacillus  and 
the  cholera  spirillum  do  not  grow  in  culture  solutions  containing 
13 


194  ACTION    OF    SALTS. 

eight  per  cent,  and  are  destroyed  by  five  hours'  exposure  to  9.23  per 
cent  (Kitasato).  Antiseptic  in  the  proportion  of  1  :  7  (Miquel). 

Potassium  Permanganate. — In  the  writer's  experiments  (1881) 
a  two-per-cent  solution  was  required  to  destroy  Micrococcus  Pasteuri 
in  the  blood  of  a  rabbit.  In  later  experiments  pus  cocci  in  bouillon 
were  killed  by  1  : 833 — time  of  exposure  two  hours.  One  per  cent 
was  found  by  Koch  not  to  destroy  anthrax  spores  in  two  days,  but 
five  per  cent  was  effective  in  one  day.  The  glanders  bacillus  is  de- 
stroyed in  two  minutes  by  a  one-per-cent  solution  (Lofner).  The 
experiments  of  Jager  show  that  a  one-per-cent  solution  is  not  reli- 
able for  the  destruction  of  anthrax  bacilli  and  other  pathogenic  bac- 
teria tested,  but  a  five-per-cent  solution  was  effective.  The  tubercle 
bacillus  was  not,  however,  killed  by  exposure  in  a  five-per-cent  solu- 
tion. According  to  Miquel,  permanganate  of  potash  is  an  antiseptic 
in  the  proportion  of  1  :  285. 

Quinine  Hydrobromate. — Antiseptic  in  the  proportion  of  1  : 182 
(Miquel). 

Quinine  Hydrochlorate. — Antiseptic  in  the  proportion  of  1  :  900 
(Ceri).  Quinine  dissolved  with  hydrochloric  acid  destroys  anthrax 
spores  in  ten  days  in  one-per-cent  solution  (Koch). 

Quinine  Sulphate. — The  writer  found  that  in  the  proportion  of 
1  :  800  quinine  prevents  the  development  of  various  micrococci  and 
bacilli.  A  ten-per-cent  solution  does  not  destroy  the  bacilli  of  symp- 
tomatic anthrax  (Arloing,  Cornevin,  and  Thomas). 

Silver  Nitrate. — Miquel  places  nitrate  of  silver  next  to  mercuric 
chloride  as  an  antiseptic,  effective  in  the  proportion  of  1  : 12,500. 
Behring  also  places  it  next  to  bichloride  as  an  antiseptic  and  germi- 
cide, and  says  that  it  is  even  superior  to  this  salt  in  albuminous 
fluids.  He  reports  that  it  prevents  the  development  of  anthrax 
spores  when  present  in  a  culture  liquid  in  the  proportion  of  1 : 80,000, 
and  in  the  proportion  of  1  : 10,000  destroys  these  spores  in  forty- 
eight  hours.  We  give  below  the  result  of  recent  experiments  by 
Boer,  in  which  the  time  of  exposure  was  two  hours  : 


Restrains 
development. 

Destroys 
vitality. 

Anthrax  bacillus  

1  :  60000 

1  :  20000 

Diphtheria  bacillus  

1  :  60000 

1  :  2500 

Glanders  bacillus  

1  :  75000 

1  :  4000 

Typhoid  bacillus  

1  :  50UOO 

1  :  4000 

Cholera  spirillum  

1  :  50000 

1  :  4000 

Silver  Chloride. — A  solution  of  chloride  of  silver  in  hyposulphite 
of  soda  is  much  less  effective  as  an  antiseptic  than  nitrate  of  silver. 


ACTION   OF   SALTS.  105 

Behring  found  that  to  prevent  the  development  of  anthrax  spores  a 
solution  of  1  :  8,000  was  required. 

Sodium  Borate. — In  the  writer's  experiments  a  saturated  solu- 
tion of  borax  was  found  to  be  without  germicidal  power.  A  twenty  - 
per-cent  solution  does  not  destroy  the  virus  of  symptomatic  anthrax 
(Arloing,  Cornevin,  and  Thomas).  A  five-per-cent  solution  failed 
to  destroy  anthrax  spores  in  fifteen  days  (Koch).  Antiseptic  in  the 
proportion  of  1  : 14  (Miquel). 

Sodium  Carbonate. — A  solution  of  2.2  per  cent  restrains  the 
growth  of  the  typhoid  bacillus,  and  of  2.47  per  cent  of  the  cholera 
spirillum.  The  first-named  bacillus  is  killed  by  four  or  five  hours' 
exposure  in  a  2.47-per-cent  solution,  and  the  cholera  spirillum  by 
3.45  per  cent  (Kitasato). 

Sodium  Chloride. — A  saturated  solution  failed  in  forty-eight 
hours  to  destroy  the  virus  of  symptomatic  anthrax  (Arloing,  Corne- 
vin,  and  Thomas).  A  saturated  solution  failed  in  forty  days  to  de- 
stroy anthrax  spores  (Koch).  A  saturated  solution  failed  in  twenty 
hours  to  destroy  the  tubercle  bacillus  in  fresh  sputum  (Schill  and 
Fischer).  In  the  writer's  experiments  a  five-per-cent  solution  failed 
to  kill  Micrococcus  Pasteuri  in  blood.  Antiseptic  in  the  proportion 
of  1  :  6  (Miquel).  According  to  Forster,  the  bacillus  of  typhoid 
fever,  the  bacillus  of  rouget,  and  the  streptococcus  of  pus  are  not 
killed  by  several  weeks'  exposure  in  strong  solutions  of  sodium  chlo- 
ride, but  the  cholera  spirillum  is  destroyed  in  a  few  hours.  Cultures 
of  the  tubercle  bacillus  are  not  sterilized  in  two  months  by  a  satu- 
rated solution ;  and  tuberculous  organs  from  an  ox,  preserved  in  a 
solution  of  salt,  did  not  lose  their  power  of  infecting  susceptible  ani- 
mals inoculated  with  material  from  the  diseased  tissue.  The  flesh 
of  swine  which  died  of  rothlauf  was  found  by  Petri  to  still  contain 
the  bacillus  in  a  living  condition  after  having  been  preserved  in 
brine  for  a  month. 

Sodium  Hyposulphite.  — In  the  writer's  experiments  a  saturated 
solution  failed  in  two  hours  to  kill  micrococci  and  bacilli.  Exposure 
for  forty-eight  hours  to  a  fifty-per-cent  solution  does  not  destroy  the 
virus  of  symptomatic  anthrax  (Arloing,  Cornevin,  and  Thomas). 
Antiseptic  in  the  proportion  of  1  :  3  (Miquel). 

Sodium  Sulphite. — The  results  with  a  saturated  solution  of  this 
salt  were,  in  the  writer's  experiments,  entirely  negative. 

Tin  Chloride. — A  one-per-cent  solution  acting  for  two  hours  de- 
stroyed the  bacteria  in  putrefying  bouillon,  while  0. 8  per  cent  failed 
(Abbott). 

Zinc  Chloride. — In  the  writer's  experiments  1:200  destroyed 
Micrococcus  Pasteuri  in  two  hours,  but  a  two-per-cent  solution  was  re- 
quired to  kill  pus  cocci  in  the  same  time  ;  spores  of  Bacillus  anthracis 


19G  ACTION   OF    SALTS. 

were  not  destroyed  by  two  hours'  exposure  in  a  ten-per-cent  solution, 
but  a  solution  of  five  per  cent  killed  the  spores  of  Bacillus  subtilis  in 
the  same  time.  Koch  found  that  anthrax  spores  germinated  after 
being  immersed  in  a  five-per-cent  solution  for  thirty  days.  The  de- 
velopment of  Bacillus  prodigiosus  is  only  slightly  retarded  by  expo- 
sure for  sixteen  hours  in  a  one-per-cent  solution.  Antiseptic  in  the 
proportion  of  1  :  520  (Miquel). 

Zinc  Sulphate. — In  the  writer's  first  experiments  a  twenty-per- 
cent solution  failed  to  destroy  in  two  hours  micrococci  obtained  from 
the  pus  of  an  acute  abscess.  In  later  experiments  a  micrococcus  from 
the  same  source  resisted  two  hours'  exposure  to  a  ten-per-cent  solu- 
tion, but  Micrococcus  tetragenus  was  destroyed  by  this  amount. 
Broken-down  beef  infusion  mixed  with  an  equal  quantity  of  a  forty  - 
per-cent  solution  was  not  sterilized  after  two  hours'  contact.  In 
Koch's  experiments  anthrax  spores  were  found  to  germinate  after 
having  been  immersed  for  ten  days  in  a  five-per-cent  solution. 


XL 

ACTION   OF   COAL-TAR  PRODUCTS,   ESSENTIAL 

OILS,   ETC. 

IN  the  present  section  we  shall  consider  the  action  upon  bacteria 
of  a  variety  of  organic  products,  and  for  convenience  will  arrange 
them  alphabetically. 

Acetone. — Anthrax  spores  grow  freely  after  two  days7  exposure 
to  the  action  of  this  agent;  at  the  end  of  five  days  their  development 
is  feeble  (Koch). 

Alcohol. — In  the  writer's  experiments  ninety-five-per-cent  alco- 
hol did  not  destroy  the  bacteria  (spores)  in  broken-down  beef  tea  in 
forty-eight  hours.  Micrococcus  Pasteuri  was  destroyed  by  two  hours' 
exposure  in  a  twenty-four-per-cent  solution  ;  pus  cocci  required  a 
forty-per-cent  solution.  Koch  found  that  absolute  alcohol  had  no 
effect  upon  anthrax  spores  exposed  to  its  action  for  one  hundred  and 
ten  days.  Schill  and  Fischer  found  that  when  tuberculous  sputum 
was  mixed  with  an  equal  amount  of  absolute  alcohol  its  infecting 
power  was  not  destroyed  in  twenty-four  hours,  but  that  in  the  pro- 
portion of  five  parts  to  one  of  sputum  it  was  effective  in  destroying 
the  tubercle  bacillus,  as  proved  by  inoculation  experiments.  Yersin 
found  that  in  pure  cultures  the  tubercle  bacillus  is  killed  by  five 
minutes'  exposure  to  the  action  of  absolute  alcohol. 

Aniline  Dyes. — Recent  researches  have  shown  that  some  of  the 
aniline  colors  possess  very  decided  germicidal  power.  Stilling  found 
that  solutions  of  methyl  violet  containing  1  : 30,000  exercise  a  re- 
straining influence  upon  the  development  of  putrefactive  bacteria 
and  pus  cocci,  and  that  these^ microorganisms  are  destroyed  by  solu- 
tions containing  1  : 2,000  to  1  : 1,000.  Methyl  violet  has  been  placed 
in  the  market  by  Merck  under  the  name  of  pyoktanin.  Janicke  re- 
ports the  following  results  with  pyoktanin  :  Staphylococcus  pyogenes 
aureus  was  restrained  in  its  development  by  solutions  containing 
1  :  2,000,000,  Bacillus  anthracis  by  1  : 1,000,000,  Staphylococcus  pyo- 
genes by  1  :  333,300,  Spirillum  cholerae  Asiaticse  by  1  :  62,500,  Bacil- 
lus typhi  abdominalis  by  1  : 5,000.  In  blood  serum  stronger  solutions 
were  required  (1 : 500,000  for  Staphylococcus  pyogenes  aureus).  Sta- 
phylococcus pyogenes  aureus,  Streptococcus  pyogenes,  and  Bacillus 
anthracis  were  killed  in  thirty  seconds  by  1  : 1,000,  the  typhoid  bacil- 


198 


ACTION   OF   COAL-TAR   PRODUCTS, 


lus  by  the  same  amount  in  thirty  minutes.  Boer  found  malachite 
green  to  be  still  more  effective  than  methyl  violet.  In  his  experi- 
ments upon  bouillon  cultures  twenty-four  hours  old,  with  two  hours' 
exposure  to  the  action  of  the  disinfectant,  he  obtained  the  following 
results  : 


MALACHITE    GREEN. 


Restrains 
development. 

Destroys 
vitality. 

1  :  120000 

1  •  40000 

Diphtheria  bacillus  

1  :  40000 

1  :8000 

Glanders  bacillus  

1  :  5000 

1  :300 

Typhoid  bacillus       .    

1  :  5000 

1  :300 

Cholera  spirillum  

1  :  100000 

1  :  5000 

METHYL   VIOLET    (PYOKTANIN). 


Restrains 
development. 

Destroys 
vitality. 

1  :  70003 

1  -5000 

Diphtheria  bacillus.  

1  :  10000 

1  •  2000 

1  :  2500 

1  :150 

Typhoid  bacillus.  •  

1  :  2500 

1  -150 

1  :  30000 

1  :  1000 

Aniline  Oil. — According  to  Riedlin,  the  addition  of  1:5  of  ani- 
line water  prevents  the  development  of  all  bacteria  in  nutrient  gelatin. 

Aromatic  Products  of  Decomposition. — Klein  has  tested  the 
germicidal  power  of  phenylpropionic  and  phenylacetic  acids.  He 
finds  that  anthrax  spores  resist  both  of  these  acids,  in  the  proportion 
of  1  : 400,  for  two  days,  but  in  the  absence  of  spores  anthrax  bacilli 
are  quickly  killed  by  a  solution  of  this  strength.  Certain  non-patho- 
genic micrococci  were  not  killed  by  exposure  for  twenty-five  minutes 
to  1  :  200.  The  caseous  matter  of  pulmonary  tuberculosis  infected 
guinea-pigs  after  exposure  for  ninety-six  hours  to  1  :  200. 

Aseptol. — A  ten-per-cent  aqueous  solution  kills  anthrax  spores  in 
ten  minutes,  and  a  three-  to  five-per-cent  solution  is  a  reliable  disin- 
fectant in  the  absence  of  spores  (Hueppe). 

Benzene,  C6H6. — Exposure  in  benzol  for  twenty  days  failed  to 
destroy  the  vitality  of  anthrax  spores  (Koch). 

Camphor. — Alcohol  saturated  with  camphor  has  no  effect  upon 
the  virus  of  symptomatic  anthrax  (Arloing,  Cornevin,  and  Thomas) 

The  experiments  of  Cadeac  and  Meunier  show  that  camphor  (oil 
of,  or  tincture?)  has  but  little  germicidal  power.  The  typhoid  ba- 


ESSENTIAL   OILS,    ETC.  199 

cillus  and  cholera  spirillum  were  only  destroyed  after  eight  to  ten 
days7  exposure  to  the  action  of  camphor  ("essence"). 

Carbolic  Acid. — Tested  upon  anthrax  spores,  Koch  found  a  one- 
per-cent  solution  to  be  without  effect  after  fifteen  days7  exposure  ;  a 
two-per-cent  solution  retarded  development  but  did  not  completely 
destroy  vitality  in  seven  days  ;  a  three-per-cent  solution  was  effec- 
tive in  two  days.  In  the  absence  of  spores  Koch  found  that  a  one- 
per-cent  solution  quickly  destroys  the  vitality  of  anthrax  bacilli. 
He  recommends  a  five-per-cent  solution  for  the  destruction  of  the 
"comma  bacillus"  in  the  discharges  of  cholera  patients,  and  a  two- 
per-cent  solution  for  the  disinfection  of  surfaces  soiled  with  such  dis- 
charges. In  the  writer's  experiments  1  :  200  destroyed  Micrococcus 
Pasteuri  in  two  hours  ;  and  pus  cocci  were  destroyed  by  1  : 125,  while 
1  : 200  failed.  Davaine  showed  by  inoculation  experiments  that  an- 
thrax bacilli  in  fresh  blood  are  destroyed  by  being  exposed  to  the 
action  of  a  one-per-cent  solution  for  one  hour.  A  two-per-cent  solu- 
tion destroys  the  dried  virus  of  symptomatic  anthrax  in  forty-eight 
hours  (Arloing,  Corneviii,  and  Thomas).  Solutions  in  oil  or  in  alco- 
hol have  been  shown  by  Koch  to  be  less  effective  than  aqueous  solu- 
tions. Thus  a  five-per-cent  solution  in  oil  failed  to  destroy  anthrax 
spores  in  one  hundred  and  ten  days,  and  the  same  solution  failed  to 
kill  the  bacilli,  in  the  absence  of  spores,  in  less  than  six  days.  A 
five-per-cent  solution  in  alcohol  did  not  destroy  anthrax  spores  in 
seventy  days.  Schill  and  Fischer  found  that  a  three-per-cent  solu- 
tion destroyed  the  infecting  power  of  tuberculous  sputum,  as  shown 
by  inoculation  into  guinea-pigs,  in  twenty-four  hours,  while  solutions 
of  one  and  two  per  cent  failed.  Bolton's  experiments  gave  the  fol- 
lowing results,  the  test  organisms  being  in  fresh  bouillon  cultures 
and  the  time  of  exposure  two  hours  :  The  cholera  spirillum,  the 
bacillus  of  typhoid  fever,  the  bacillus  of  schweinerothlauf,  Brieger's 
bacillus,  the  bacillus  of  green  pus,  and  the  pus  cocci  (Staphylococcus 
pyogenes  aureus,  albus,  and  citreus,  and  Streptococcus  pyogenes) 
were  all  killed  by  a  solution  of  one  per  cent,  while  in  a  majority  of 
the  experiments  a  one-half -per-cent  (1  : 200)  solution  failed.  Cul- 
tures of  the  typhoid  bacillus  in  flesh-peptone-gelatin  gave  the  same 
result  (1  : 100  with  two  hours7  exposure),  and  the  addition  of  ten  per 
cent  of  dried  egg  albumin  to  bouillon  cultures  did  not  influence  the 
result. 

The  experiments  of  La  Place  show  that  the  addition  of  hydro- 
chloric acid  to  a  disinfecting  solution  containing  carbolic  acid  greatly 
increases  its  germicidal  power  for  spores.  Thus  it  is  stated  that 
"  two  per  cent  of  crude  carbolic  acid  with  one  per  cent  of  pure  hydro- 
chloric acid  destroyed  anthrax  spores  in  seven  days,  while  two  per 
cent  of  carbolic  acid  or  one  per  cent  of  hydrochloric  acid  alone  did 


200  ACTION   OF   COAL-TAR   PRODUCTS, 

not  destroy  these  spores  in  thirty  days.  A  f our-per-cent  solution  of 
crude  carbolic  acid  with  two  per  cent  of  hydrochloric  acid  destroyed 
spores  in  less  than  an  hour  ;  four  per  cent  of  carbolic  acid  alone  did 
not  destroy  them  in  twelve  days.  Van  Ermengem  reports  that  in 
his  experiments  the  cholera  spirillum  in  chicken  bouillon  was  killed 
in  less  than  half  an  hour  by  1  :  600,  and  that  in  blood  serum  1  :  400 
was  effective.  Nicati  and  Rietsch  fix  the  germicidal  power  for  the 
cholera  spirillum  as  1  : 200,  the  time  of  exposure  being  ten  minutes  ; 
Ramon  and  Cajal,  1  :  50.  Boer  gives  the  following  results,  the  time 
of  exposure  being  two  hours,  cultures  in  bouillon  twenty-four  hours 
old  : 


Restrains 
development. 

Destroys 
vitality. 

1  -750 

1  -300 

1  -500 

1  -300 

1  :500 

1  :300 

Typhoid  bacillus  

1  :400 

1  :200 

Cholera  spirillum  

1  :600 

1  :400 

Leitz  reports  the  following  results  :  The  dejections  of  patients 
suffering  from  typhoid  fever,  mixed  in  equal  quantity  with  the  disin- 
fecting solution,  were  sterilized  by  a  five-per-cent  solution  of  car- 
bolic acid  in  three  days.  Pure  cultures  of  the  typhoid  bacillus  were 
sterilized  in  fifteen  minutes  by  a  five-per-cent  solution. 

In  the  experiments  of  Nocht  upon  anthrax  spores  it  was  found 
that  while  at  the  room  temperature  these  spores  were  not  destroyed 
by  several  days'  exposure  in  a  five-per-cent  solution,  they  were  de- 
stroyed in  three  hours  by  the  same  solution  at  a  temperature  of  37.5°. 

Carbolic  acid  prevents  putrefactive  changes  in  bouillon  when  pre- 
sent in  the  proportion  of  1  :  333  (Miquel).  The  tubercle  bacillus  is 
killed  in  thirty  seconds  by  a  five-per-cent  solution,  and  in  one  minute 
by  a  one-per-cent  solution  (Yersin). 

Coffee  Infusion. — Experiments  have  been  made  by  Heim  and  by 
Liideritz  on  the  antiseptic  power  of  an  infusion  of  coffee.  The  first- 
named  author  found  that  anthrax  bacilli  no  longer  developed  after 
three  hours'  exposure  in  a  ten-per-cent  solution,  but  spores  were  not 
killed  at  the  end  of  a  week.  Streptococci  in  a  bouillon  culture  re- 
quired twenty-four  hours'  exposure,  and  the  staphylococci  of  pus  were 
not  destroyed  in  this  time.  Ltideritz  found  that  a  three-per-cent  in- 
fusion restrained  the  growth  in  nutrient  gelatin  of  the  typhoid  ba- 
cillus, and  a  five-per-cent  infusion  killed  the  bacillus  in  two  days  ; 
the  cholera  spirillum  failed  to  grow  in  presence  of  one  per  cent,  and 
a  solution  of  this  strength  killed  it  in  seven  hours  ;  Staphylococcus 


ESSENTIAL,  OILS,    ETC.  .    201 

pyogenes  aureus  was  prevented  from  developing  by  two  per  cent, 
and  was  killed  in  six  days  by  a  five-per-cent  solution  ;  Streptococcus 
pyogenes  was  prevented  from  growing  by  one  per  cent,  and  killed  by 
a  ten-per-cent  solution  in  one  day  ;  Proteus  vulgaris  did  not  grow  in 
presence  of  2. 5  per  cent,  and  was  killed  in  two  days  by  ten  per  cent. 
The  question  as  to  what  constituent  of  the  infusion  of  roasted  coffee 
was  the  active  germicidal  agent  was  not  determined,  but  the  authors 
referred  to  agree  that  it  was  not  caffeine. 

Creolin. — This  is  a  coal-tar  product  which  resembles  crude  carbolic 
acid  in  appearance,  but  smells  rather  like  tar  than  like  phenol.  It 
makes  a  milky  emulsion  with  water,  which  has  been  proved  by  nu- 
merous experiments  to  possess  very  decided  germicidal  power,  being 
superior  to  carbolic  acid.  The  first  careful  test  of  the  germicidal 
power  of  this  agent  was  made  by  Esmarch,  who  found  that  a  solu- 
tion of  1  :  200  killed  the  cholera  spirillum  in  a  minute,  the  typhoid 
bacillus  at  the  end  of  several  days.  Anthrax  spores  were  not  de- 
stroyed in  twenty  days  by  a  five-per-cent  solution,  but  this  solution 
killed  the  tubercle  bacillus  attached  to  silk  threads  which  were  im- 
mersed in  it  for  a  short  time,  and  also  disinfected  tuberculous  sputum. 
Behring  has  shown  that  in  albuminous  liquids  creolin  is  less  effective 
than  carbolic  acid.  In  blood  serum  1  : 175  was  required  to  restrain 
the  development  of  staphylococci,  and  1  : 100  to  destroy  the  same  in 
ten  minutes.  Van  Ermengem,  as  a  result  of  numerous  experiments, 
arrived  at  the  conclusion  that  creolin  is  a  cheap  and  useful  disinfect- 
ing agent,  in  a  five-per-cent  solution,  for  various  pathogenic  organ- 
isms. Kaupe  reports  that  in  his  experiments  a  ten-per-cent  solution 
killed  anthrax  spores  in  twenty-four  hours.  According  to  Boer,  a 
solution  of  1  : 5,000  destroys  anthrax  bacilli  in  bouillon  cultures  in 
two  hours,  1  : 2,000  diphtheria  bacilli,  1  :  300  the  glanders  bacillus, 
1 : 250  the  typhoid  bacillus,  and  1  : 3,000  the  cholera  spirillum. 

Creosote. — This  agent  was  found  by  the  writer  to  be  fatal  to 
micrococci  in  the  proportion  of  1  :  200.  In  the  proportion  of  one  per 
cent  it  failed,  after  twenty  hours5  exposure,  to  destroy  tubercle  ba- 
cilli in  sputum  (Schill  and  Fischer).  A  saturated  aqueous  solution 
does  not  destroy  the  tubercle  bacillus  in  cultures  in  twelve  hours 
(Yersin).  Guttman,  in  extended  experiments  upon  various  patho- 
genic organisms,  found  that  development  was  prevented  by  1  :  3,000 
to  1  :  4,000.  A  solution  containing  1  : 300  killed  Bacillus  pyocyanus 
and  Bacillus  anthracis  in  one  minute,  Bacillus  prodigiosus  in  two 
minutes,  and  the  Finkler-Prior  spirillum  in  one  minute  in  the  pro- 
portion of  1  : 600. 

Cresol. — This  is  a  dark,  reddish-brown,  transparent  fluid,  some- 
what thinner  than  creolin,  and,  like  it,  having  an  odor  of  tar.  It 
forms  an  emulsion  with  water,  which  is  not  so  stable  as  that  formed 


202  ACTION   OF   COAL-TAR   PRODUCTS, 

by  creolin.  Of  the  three  cresols,  ortho-,  meta-,  and  paracresol,  the 
second  was  found  by  Frankel  to  be  most  active.  This  author  states 
that  the  addition  of  sulphuric  acid  adds  greatly  to  its  germicidal 
power.  A  four-per-cent  solution,  containing  equal  parts  of  cresol 
and  H2S04,  killed  anthrax  spores  in  less  than  twenty-four  hours.  In 
Behring's  experiments  a  solution  containing  ten  per  cent  of  each  killed 
anthrax  spores  in  eighty  minutes,  and  five  per  cent  of  each  in  one 
hundred  minutes,  while  an  eighteen-per-cent  solution  of  sulphuric 
acid  alone  did  not  kill  them  in  twenty  four  hours.  In  the  experi- 
ments of  Jager  a  two-per-cent  solution  destroyed  the  tubercle  bacillus 
in  cultures  and  in  sputum.  As  a  result  of  his  experiments  Behring 
concludes  that  cresol  has  no  advantage  over  carbolic  acid  as  a  ger- 
micide for  the  destruction  of  spores.  Tested  upon  Staphylococcus 
aureus,  Streptococcus  erysipelatos,  and  Bacillus  pyocyanus,  Frankel 
found  that  a  solution  of  0. 3  per  cent  destroyed  these  microorganisms 
in  five  minutes,  while  a  two-per-cent  solution  of  carbolic  acid  re- 
quired fifteen  minutes'  contact  to  accomplish  the  same  result. 

Trikresol  (Schering)  has  been  tested,  with  favorable  results,  by 
several  bacteriologists.  According  to  Hammerl  it  is  about  twice  as 
active  a  germicide  as  carbolic  acid. 

Diaphtherin  (oxychinaseptol)  has  considerable  antiseptic  power, 
as  shown  by  the  experiments  of  Rohrer  and  others.  Two  to  four 
drops  of  a  one-per-cent  solution  was  found  to  prevent  the  develop- 
ment of  test  organisms  (Staphylococcus  pyogenes  aureus  and  Bacillus 
anthracis)  in  twelve  cubic  centimetres  of  bouillon.  Stable  (1893) 
also  finds  that  as  an  antiseptic  it  is  far  superior  to  carbolic  acid  or  lysol, 
and  that  it  has  the  advantage  of  being  non-toxic.  Tested  upon  an- 
thrax spores  it  was  found  to  be  comparatively  inactive  as  a  germicide. 
A  fifteen-per-cent  solution  destroyed  anthrax  spores  in  three  days. 

Disinfektol. — This  is  a  coal-tar  product  similar  to  creolin  which 
has  been  recommended  in  Germany  for  disinfecting  purposes.  It  is 
an  oily,  dark-brown  fluid  having  a  specific  gravity  of  1.086.  It  forms 
an  emulsion  with  water,  which  has  a  slightly  alkaline  reaction.  It 
has  been  tested  upon  typhoid  stools  by  Uffelmann  and  by  Beselin. 
The  last-named  author  gives  the  following  summary  of  the  results 
obtained  :  An  emulsion  of  five  per  cent  of  disinf  ektol  equals  in  value, 
for  the  disinfection  of  the  liquid  discharges  of  typhoid  patients,  12.5 
per  cent  of  creolin,  thirty-three  per  Cent  of  hydrochloric  acid,  five  per 
cent  of  carbolic  acid,  1  : 500  of  mercuric  chloride. 

Ether. — Anthrax  spores  may  germinate  after  being  immersed  in 
sulphuric  ether  for  eight  days  (Koch).  The  tubercle  bacillus  is  de- 
stroyed by  ten  minutes'  exposure  to  the  action  of  ether  (Yersin). 

Essential  Oils. — Chamberlain  has  made  an  extended  series  of 
experiments  to  determine  the  antiseptic  power  of  the  vapor  of  vola- 


ESSENTIAL   OILS,    ETC.  203 

tile  oils.  A  large  number  of  essential  oils  tested  were  found  to  pre- 
vent the  development  of  the  anthrax  bacillus,  while  a  few  did  not. 
At  the  end  of  six  days  the  tubes  were  opened  and  the  oil  absorbed  by 
the  culture  liquid  allowed  to  evaporate.  Cultures  were  now  obtained 
from  all  except  the  following,  which,  it  was  inferred,  had  destroyed 
the  vitality  of  the  spores  :  Angelica,  cinnamon  of  China,  cinnamon 
of  Ceylon,  geranium  of  France,  geranium  of  Algeria,  origanum. 

Cadeac  and  Meunier  have  also  made  extended  experiments  upon 
the  typhoid  bacillus  and  the  bacillus  of  glanders,  for  the  purpose  of 
determining  the  germicidal  power  of  agents  of  this  class.  Their 
method  consisted  in  the  introduction  of  a  sterilized  platinum  needle 
into  a  pure  culture  of  the  test  organism,  in  immersing  it  in  the 
essential  oil  for  a  certain  time,  and  then  making  with  it  a  puncture 
in  a  suitable  solid  culture  medium.  Their  results  are  given  belovr 
for  the  typhoid  bacillus. 

Essences  which  kill  the  bacillus  after  a  contact  of  less  than 
twenty-four  hours : 

At  the  end  of— 

Cinnamon  of  Ceylon,       .  .  .  .  .12  minutes. 

Cloves,  .  .  .  .  .25 

Eug-enol,    .......       30 

Thyme,  ......  35 

Wild  thyme 35 

Verbena  of  India,       .....  45 

Geranium  of  France,        .  .  .  .  .50 

Origanum,       ......  75 

Patchouly,  ......       80 

Zedoary,  ......  2  hours. 

Absinthe,  .  .  .  .  .  .  4      " 

Sandal  wood,    .  .  .  .  .  .  12      " 

The  following  were  effective  in  from  twenty-four  to  forty-eight 
hours:  Cumin,  caraway,  juniper,  matico,  galbanum,  valerian,  citron, 
angelica,  celery,  savin,  copaiba,  pepper,  turpentine,  opopanax,  rose, 
chamomile  ;  the  following  required  from  two  to  four  days :  Illicium, 
sassafras,  tuberose,  coriander;  the  following  from  four  to  eight  days: 
Calamus,  sage,  fennel,  mace,  cascarilla,  orange  of  Portugal;  the  fol- 
lowing in  eight  to  ten  days  :  Mint,  nutmeg,  rosemary,  carrot,  mus- 
tard, anise,  onion,  marjoram,  bitter  almonds,  cherry  laurel,  myrtle, 
lavender,  eucalyptus,  cedar,  cajuput,  winter  green,  camphor. 

Biedlin  reports  as  the  result  of  his  experiments  that  the  essential 
oils  which  have  the  greatest  antiseptic  value  are  oil  of  lavender,  eu- 
calyptus, rosemary,  and  cloves. 

Eucalyptol. — Chabaunes  and  Ferret  found  that  a  five-per-cent 
solution  of  eucalyptol  is  without  effect  upon  tubercle  bacilli  in  spu- 
tum. According  to  Behring,  eucalyptol  is  about  four  times  less  ac- 
tive as  a  disinfectant  than  carbolic  acid. 


204  ACTION    OF   COAL-TAR   PRODUCTS, 

Euphorin  (Phenylurethan)  has  been  tested  by  Colasanti  (1894), 
who  finds  that  it  has  rather  feeble  germicidal  activity. 

Formaldehyde  (formol,  formalin)  has  very  decided  germicidal 
power.  According  to  Pottevin  (1894)  in  the  absence  of  spores  a  solu- 
tion of  1 : 1,000  kills  bacteria,  in  comparatively  small  numbers,  in  from 
fifteen  minutes  to  several  hours.  For  the  destruction  of  spores  a 
much  stronger  solution  is  required — a  fifteeii-per-cent  solution  at 
15°  C.  killed  anthrax  spores  in  one  and  one-half  hours,  and  spores 
of  Bacillus  subtilis  in  twenty  hours.  At  higher  temperatures  the 
germicidal  action  is  more  energetic,  and  microorganisms  exposed  to 
the  vapor  of  formol  are  very  quickly  destroyed.  Vanderlinden  and 
de  Buck  (1895)  find  that  solutions  of  formalin  are  decidedly  inferior 
to  corresponding  solutions  of  carbolic  acid,  creolin,  or  solveol,  and  are 
too  irritating  to  be  used  in  surgical  practice.  They  report  that  a 
solution  of  five  per  cent  failed  to  destroy  their  test  organisms- 
Bacillus  coli  communis,  Bacillus  typhi  abdominalis,  Staphylococcus 
pyogenes  aureus.  Experiments  made  by  Reed,  at  the  Army  Medical 
Museum  in  Washington,  show  that  the  diphtheria  bacillus  and  other 
test  organisms  are  quickly  killed  by  formalin  vapor. 

Glycerin  has  no  action  upon  the  virus  of  symptomatic  anthrax 
(Arloing,  Cornevin,  and  Thomas),  and  is  inert  as  regards  the  spores 
of  anthrax  (Koch).  Glycerin  prevents  putrefactive  decomposition  in 
bouillon  when  present  in  the  proportion  of  1  :-4  (Miquel).  Roux  has 
shown  that  the  addition  of  five  per  cent  of  glycerin  to  a  culture 
medium  is  favorable  to  the  growth  of  the  tubercle  bacillus;  it  is  also 
appropriated  as  pabulum  by  various  other  species. 

Guaiacol. — Kuprianow,  as  a  result  of  extended  experiments  with 
this  agent  (1894),  reports  that  it  ranks  below  cresol  and  carbolic  acid 
as  a  germicide.  In  the  proportion  of  1 :  500  it  restrains  the  develop- 
ment of  the  cholera  spirillum,  and  the  author  named  suggests  its  in- 
ternal administration  in  this  disease  on  account  of  its  non-toxic  and 
non-irritant  properties. 

Hydroxylamin. — Heinisch  found  that  the  development  of  the 
anthrax  bacillus  is  prevented  by  1 : 77  of  hydroxylamin  hydro- 
chlorate,  and  of  the  diphtheria  bacillus  by  1 :  75.  In  these  experiments 
a  solution  of  soda  was  added  to  release  the  hydroxylamin.  Marp- 
mann  found  that  1 : 100  preserved  milk  without  change  for  four 
to  six  weeks,  and  that  alkaline  fermentation  of  urine  was  prevented 
by  1:1,000. 

IchthyoL — Latteux  (1892)  reports  that  the  various  pathogenic 
bacteria  used  by  him  as  test  organisms  were  killed  by  a  five-per-cent 
solution  (time  •?)  with  exception  of  Streptococcus  pyogenes,  which 
required  a  six  to  seven-per-cent  solution.  The  more  recent  experi- 
ments of  Abel  (1893)  gave  less  favorable  result,  but  the  agent  was 


ESSENTIAL  OILS,    ETC.  205 

shown  to  have  considerable  antiseptic  value — 1 :  2,000  restrained  the 
development  of  streptococci;  1 :  500  of  the  diphtheria  bacillus;  1 :  20 
of  Staphylococeus  pyogenes  aureus;  1:33  the  bacillus  of  typhoid 
fever.  Streptococci  and  diphtheria  bacilli  were  destroyed  in  twenty- 
four  hours  by  a  solution  of  1 :  200;  Staphylococcus  aureus,  subjected 
to  the  action  of  pure  ichthyol,  was  destroyed  in  five  hours — in  a  five- 
per-cent  solution  it  survived  for  four  days.  Cultures  of  the  typhoid 
bacillus  mixed  with  a  fifty-per-cent  solution  were  not  completely 
•sterilized  in  thirty  hours;  a  small  number  of  bacilli  in  bouillon  were, 
however,  destroyed  by  a  three-per-cent  solution  in  forty-eight  hours. 
Anthrax  spores  on  silk  threads  were  not  destroyed  by  a  fifty-per-cent 
solution  at  the  end  of  one  hundred  and  forty  days. 

Indol. — When  added  in  excess  to  water  this  agent  failed  to  de- 
stroy anthrax  spores  in  eighty  days  (Koch). 

Izal  is  a  coal-tar  product  which  has  recently  been  introduced  as 
a  disinfectant.  Klein  (1892)  reports  that  in  the  strength  of  ten  per 
cent  it  kills  anthrax  spores  in  fifteen  minutes.  In  the  absence  of 
spores  various  pathogenic  bacteria  were  killed  in  five  minutes  by  a 
solution  containing  1 :  200. 

Lanolin. — According  to  Gottstein,  various  microorganisms  tested 
by  him  failed  to  grow  in  cultures  after  having  been  in  contact  with 
pure  lanolin  for  five  to  seven  days. 

Loretin. — Korff  (1895)  claims  for  this  agent  that  a  two-per-cent 
solution  is  superior  to  corresponding  solutions  of  lysol,  metakresol, 
or  phenol,  and  that  it  has  the  advantage  of  being  non-toxic,  odorless, 
and  non-irritating. 

Lysol. — Weiss  (1895)  has  tested  this  product  and  reports  that  a 
solution  of  three-fourths  per  cent  destroyed  his  test  organisms  (pus 
cocci,  typhoid  bacillus,  Bacillus  coli  communis,  etc.)  in  five  minutes. 
Anthrax  spores  were  destroyed  by  the  same  solution  in  one  hour. 

Naphthol . — In  the  proportion  of  1 :  10,000  naphthol  prevents  the 
development  of  the  glanders  bacillus,  the  anthrax  bacillus,  the  typhoid 
bacillus,  the  micrococcus  of  fowl  cholera,  of  Staphylococcus  aureus 
and  albus,  and  of  several  other  microorganisms  tested  by  Maximo- 
vitch.  The  same  author  states  that  although  insoluble  in  cold  water, 
water  at  70°  C.  dissolves  0.44  in  one  thousand  parts.  When  urine  is 
shaken  up  with  naphthol  in  powder  it  does  not  undergo  fermenta- 
tion. 

In  the  experiments  of  Foote  hydronaphthol  was  found  to  show 
some  germicidal  power  in  the  proportion  of  1 :  2,300,  but  the  conclu- 
sion is  reached  that  a  saturated  aqueous  solution  (1 :  1,150)  does  not 
equal  a  one-per-cent  solution  of  carbolic  acid  or  of  creolin. 

The  writer,  in  1892,  obtained  the  following  results  in  experiments 
with  naphthols  upon  the  cholera  spirillum. 


206  ACTION   OF   COAL-TAR   PRODUCTS, 

Alpha-naphthol  and  beta-naplitliol  have  about  the  same  antiseptic  and 
germicidal  value.  In  the  proportion  of  1  : 16,000  both  prevent  the  develop- 
ment of  the  cholera  spirillum  in  peptonized  beef -tea,  while  1  :  24, 000  fails  to 
prevent  development.  In  the  proportion  of  1  :  3,000  both  destroy  the  vital- 
ity of  the  cholera  spirillum  in  bouillon  cultures,  twenty-four  hours  old, 
after  two  hours'  contact,  while  1 :  4,000  fails  to  destroy  this  microorganism 
in  the  time  mentioned — two  hours. 

In  experiments  made  with  a  solution  of  1:1,000,  added  to  an  equal 
quantity  of  a  twenty-four  hours  old  bouillon  culture — making-  1  :  2,000  after 
mixture — and  in  which  the  time  of  contact  varied  from  five  to  thirty  minutes, 
alpha-,  beta-,  and  hydronaphthol  were  found  to  destroy  the  cholera  germ  by 
fifteen  minutes'  exposure,  but  to  fail  after  ten  minutes'  contact,  so  that  the 
germicidal  value  of  each  of  these  is  similar,  or  nearly  so. 

In  all  these  experiments  the  line  was  sharply  drawn  between  success  and 
failure.  No  development  occurred  and  the  bouillon  remained  transparent 
in  those  experiments  in  which  the  germicidal  action  was  complete,  and  a 
characteristic  development  occurred  within  twenty -four  hours  in  those  ex- 
periments in  which  there  was  a  failure  to  destroy  the  spirillum. 

Benzo-naphthol  has  no  germicidal  power,  probably  because  it  is  insoluble 
in  water.  At  least  this  is  my  inference  from  the  experiments  made.  One 
gamme  was  added  to  one  thousand  cubic  centimetres  of  distilled  water,  and 
after  vigorous  shaking  was  placed  in  the  steam  sterilizer  for  half  an  hour. 
At  the  end  of  this  time  the  greater  portion,  at  least,  of  the  benzo-naphthol  re- 
mained undissolved  at  the  bottom  of  the  flask.  The  saturated  solution  (?) 
was  then  filtered  and  added  to  recent  bouillon  cultures  of  the  cholera  spiril- 
lum in  the  proportion  of  1 : 1,  1:2,  1 :  4,  and  2:1.  At  the  end  of  two  hours 
sterile  bouillon  in  test  tubes  was  inoculated  from  each  of  these  and  placed  in 
the  incubating  oven.  At  the  end  of  forty-eight  hours  a  characteristic  devel- 
opment of  the  cholera  spirillum  had  occurred  in  all  of  the  tubes. 

Olive  Oil. — Anthrax  spores  germinate  after  having  been  im- 
mersed for  ninety  days  in  pure  olive  oil  (Koch). 

Oil  of  Mustard. — Koch  found  that  the  development  of  anthrax 
spores  is  prevented  by  1 : 33,000. 

Oil  of  Peppermint. — A  five-per-cent  solution  in  alcohol  failed  in 
twelve  days  to  destroy  anthrax  spores,  but  the  development  of  these 
spores  is  restrained  by  1 :  33,000  (Koch). 

Oil  of  Turpentine  destroys  anthrax  spores  in  five  days,  but  failed 
to  do  so  in  one  day  (Koch).  The  development  of  anthrax  spores  is 
prevented  by  1:  75,000  (Koch).  The  addition  of  1 :  200  to  nutrient 
gelatin  prevents  the  development  of  bacteria  (Riedlin) .  An  excess 
of  oil  of  turpentine  added  to  a  liquefied  gelatin  culture  of  Staphylo- 
coccus  aureus  does  not  destroy  this  micrococcus  in  five  hours  (v. 
Christmas-Dirckinck-Holmfeld). 

Saprol. — Laser  (189*2)  recommends  tbis  agent  for  the  disinfection 
of  the  excreta  of  cholera  and  typhoid  patients.  He  reports  that  in  the 
proportion  of  1 :  100  it  sterilizes  liquid  fa3ces  in  twenty-four  hours. 

Skatol  in  excess  in  water  has  no  germicidal  power,  as  tested  upon 
anthrax  spores  (Koch). 

Smoke. — The  researches  of  Beu  show  that  meats  which  have  been 
preserved  by  smoking  commonly  contain  living  bacteria  capable  of 
growing  in  culture  media;  and  Petri  has  shown  that  pork  which  has 


ESSENTIAL   OILS,    ETC.  207 

been  salted  for  a  month  and  then  smoked  for  fourteen  days  may  still 
contain  the  bacillus  of  rothlauf  in  a  living  condition,  as  shown  by  in- 
oculation experiments.  It  was  not  until  about  six  months  after  smok- 
ing that  the  bacillus  failed  to  give  evidence  of  vitality. 

Thymol. — A  five-per-cent  solution  in  alcohol  does  not  destroy 
anthrax  spores  in  fifteen  days,  but  the  development  of  these  spores 
is  retarded  by  a  solution  of  1  : 80, 000  (Koch).  The  anthrax  bacillus 
and  staphylococci  fail  to  grow  in  culture  media  containing  1  :  3,000 
(Samter).  The  tubercle  bacillus  is  destroyed  by  contact  with  thy- 
mol for  three  hours  (Yersin).  Thymol  has  about  four  times  less 
germicidal  power  than  carbolic  acid  (Behring).  Antiseptic  in  the 
proportion  of  1  : 1,340  (Miquel). 

Tobacco  Smoke. — Tassinari  found  that  tobacco  smoke  restrains 
the  development  of  bacteria,  and  that  certain  species  failed  to  de- 
velop after  exposure  for  half  an  hour  in  an  atmosphere  of  tobacco 
smoke — spirillum  of  cholera  and  Friedlander's  bacillus. 


XII. 

ACTION  OF  BLOOD   SERUM  AND   OTHER  ORGANIC 

LIQUIDS. 

Blood  Serum. — Bacteriologists  have  long  been  aware  of  the  fact 
that  many  species  of  bacteria,  when  injected  into  the  circulation  of  a, 
living  animal,  soon  disappear  from  the  blood,  and  that  the  blood  of 
such  an  animal  a  few  hours  after  an  injection  of  putrefactive  bacte- 
ria, for  example,  does  not  contain  living  bacteria  capable  of  develop- 
ing in -a  suitable  nutrient  medium.  Wyssokowitsch,  in  an  extended 
series  of  experiments,  has  shown  that  non-pathogenic  bacteria  in- 
jected into  the  circulation  may  be  obtained  in  cultures  from  the  liver, 
spleen,  kidneys,  and  bone  marrow  after  they  have  disappeared  from 
the  blood,  but  that,  as  a  rule,  those  present  in  these  organs  have  lost 
their  vitality,  as  shown  by  culture  experiments,  in  a  period  varying 
from  a  few  hours  to  two  or  three  days.  According  to  the  theory  of 
Metschnikoff ,  this  destruction  of  bacteria  in  the  blood  and  tissues  of  a 
living  animal  is  effected  by  the  cellular  elements,  and  especially  by 
the  leucocytes,  which  pick  up  and  digest  these  vegetable  cells  very 
much  as  an  amoeba  disposes  of  similar  microorganisms  which  serve 
it  as  food.  Some  such  theory  seemed  necessary  to  account  for  the 
disappearance  of  bacteria  from  the  blood  before  the  demonstration 
was  made  that  the  serum  of  the  circulating  fluid,  quite  indepen- 
dently of  its  cellular  elements,  possesses  very  decided  germicidal 
power. 

Yon  Fodor  first  (1887)  called  attention  to  the  fact  that  anthrax  ba- 
cilli maybe  destroyed  by  freshly  drawn  blood  ;  and  Nuttall  (1888), 
in  an  extended  series  of  experiments,  showed  that  various  bacteria 
are  destroyed  within  a  short  time  by  the  fresh  blood  of  warm- 
blooded animals.  Thus  the  anthrax  bacillus  in  rabbit's  blood  was 
usually  killed  in  from  two  to  four  hours  when  the  temperature  was 
maintained  at  37°-38°  C.,  and  the  same  result  was  obtained  with 
pigeon's  blood  at  41°  C.  But  when  the  blood  was  allowed  to  stand 
for  a  considerable  time,  or  was  heated  for  forty-five  minutes  to 
45°  C.,  it  served  as  a  culture  fluid,  and  an  abundant  development  of 
anthrax  bacilli  occurred  in  it.  Bacillus  subtilis  and  Bacillus  mega- 


ACTION    OF   BLOOD    SERUM    AND    OTHER    ORGANIC    LIQUIDS.     209 

therium  were  also  destroyed  in  two  hours  by  fresh  rabbit's  blood, 
but  it  was  without  action  on  Staphylococcus  pyogenes  aureus,  which 
at  a  temperature  of  37.5°  C.  was  found  to  have  increased  in  num- 
bers at  the  end  of  two  hours.  Further  researches  by  Nissen  and 
Behring  show  that  there  is  a  wide  difference  in  the  blood  of  dif- 
ferent animals  as  to  germicidal  power,  and  that  certain  bacteria 
are  promptly  destroyed,  while  other  species  are  simply  restrained  for 
a  time  in  their  development  or  are  not  affected.  Thus  Nissen  found 
that  the  cholera  spirillum,  the  bacillus  of  anthrax,  the  bacillus  of 
typhoid  fever,  and  Friedlander's  pneumococcus  were  killed,  while 
Staphylococcus  pyogenes  aureus  and  albus,  the  streptococcus  of  ery- 
sipelas, the  bacillus  of  fowl  cholera,  the  bacillus  of  rothlauf,  and 
Proteus  hominis  were  able  to  multiply  in  rabbit's  blood  after  having 
been  restrained  for  a  short  time  in  their  development.  In  the  case 
of  the  cholera  spirillum  a  period  of  ten  to  forty  minutes  sufficed  for 
the  complete  destruction  of  a  limited  number,  but  when  the  number 
exceeded  1,200,000  per  cubic  centimetre  they  were  no  longer  de- 
stroyed with  certainty,  and  after  five  hours  an  increase  occurred. 
The  anthrax  bacillus  was  commonly  destroyed  within  twenty  minutes 
and  the  typhoid  bacillus  at  the  end  of  two  hours.  In  the  experi- 
ments of  Bearing  and  Mssen  it  was  found  that  the  most  pronounced 
germicidal  effect  upon  the  anthrax  bacillus  was  obtained  from  the 
blood  of  the  rat,  an  animal  which  has  a  natural  immunity  against 
anthrax ;  while  the  blood  of  the  guinea-pig,  a  very  susceptible  ani- 
mal, had  no  restraining  effect  and  served  as  a  favorable  culture 
medium  for  the  anthrax  bacillus.  And  the  remarkable  fact  was  de- 
monstrated that  when  the  blood  of  a  rat  was  added  to  the  blood  of 
the  guinea-pig  in  the  proportion  of  1:8,  it  exercised  a  decided  re- 
straining influence  upon  the  growth  of  the  anthrax  bacillus.  Later 
researches  have  shown  that  cultivation  in  the  blood  of  an  immune 
animal  causes  an  attenuation  of  the  virulence  of  an  anthrax  cul- 
ture (Ogata  and  Jasuhara)  ;  and  also  that  the  injection  of  the  blood 
of  a  frog  or  rat — naturally  immune — into  a  susceptible  animal  which 
has  been  inoculated  with  a  virulent  culture  of  the  anthrax  bacillus, 
will  prevent  the  death  of  the  inoculated  animal. 

Buchner  has  shown  that  the  germicidal  power  of  the  blood  of 
dogs  and  rabbits  does  not  depend  upon  the  presence  of  the  cellular 
elements,  but  is  present  in  clear  serum  which  has  been  allowed  to 
separate  from  the  clot  in  a  cool  place.  Exposure  for  an  hour  to  a 
temperature  of  55°  C.  destroys  the  germicidal  action  of  serum  as 
well  as  of  blood  ;  the  same  effect  is  produced  by  heating  to  52°  C.  for 
six  hours  or  to  45. 6°  C.  for  twenty  hours.  The  germicidal  power 
of  blood  serum  is  not  destroyed  by  freezing  and  thawing,  but  is 
lost  after  it  has  been  kept  for  some  time.  Buchner's  experiments  led 
14 


ACTION   OF   BLOOD    SERUM   AND    OTHER   ORGANIC   LIQUIDS. 

him  to  the  conclusion  that  the  germicidal  power  of  fresh  blood 
serum  depends  upon  the  presence  of  some  albuminous  body  present 
in  it.  This  view  is  sustained  by  the  researches  of  Ogata,  who  has 
obtained  from  the  blood  of  dogs  and  other  animals  a  glycerin  ex- 
tract of  a  "ferment"  which  is  insoluble  in  alcohol  or  in  ether  and 
which  has  germicidal  properties. 

According  to  Emmerich  and  Tsuboi  (1893),  when  the  serum- 
albumin  is  precipitated  by  alcohol,  dried  in  a  vacuum  at  40°  C.,  and 
dissolved  in  water  it  has  no  longer  any  germicidal  activity.  But  if 
the  precipitated  and  dried  albumin  is  dissolved  at  3(J°  C.  in  a  weak 
solution  (0.05-0.08  per  cent)  of  soda  or  potash  it  recovers  its  original 
germicidal  value. 

It  has  been  demonstrated  by  several  experimenters  that  other 
albuminous  fluids  possess  a  similar  germicidal  power.  Thus  Nuttall 
found  that  a  pleuritic  exudation  from  man  destroyed  the  anthrax 
bacillus  in  an  hour,  the  aqueous  humor  of  a  rabbit  in  two  hours. 
Wurz  has  experimented  with  fresh  egg  albumin,  and  found  that  the 
anthrax  bacillus  failed  to  grow  after  having  been  exposed  for  an  hour 
to  the  action  of  albumin  from  a  hen's  egg ;  other  bacteria  tested 
were  not  killed  so  promptly,  but  a  decided  germicidal  action  was 
manifested.  Prudden  has  shown  that  the  albuminous  fluid  obtained 
from  a  hydrocele,  or  from  the  abdominal  cavity  in  ascites,  possesses 
similar  germicidal  power  ;  and  Fokker  has  demonstrated  that  fresh 
milk  destroys  the  vitality  of  certain  bacteria  which  induce  an  acid 
fermentation  of  this  fluid. 

The  results  heretofore  referred  to  induced  Hankin  to  experiment 
with  cell  globulin  obtained  from  the  spleen  or  lymphatic  glands  of  a 
dog  or  cat.  This  is  extracted  by  means  of  a  solution  of  chloride  of 
sodium,  the  solution  is  filtered,  and  the  globulin  precipitated  by  the 
addition  of  alcohol.  The  precipitate  is  washed  and  again  dissolved 
in  salt  solution.  The  result  showed  that  this  cell  globulin  possesses 
germicidal  power  similar  to  that  of  blood  serum. 

Mucus. — The  experiments  of  Wurtz  and  Lermoyez  (1893)  show 
that  nasal  mucus  has  germicidal  properties,  especially  for  the  anthrax 
bacillus.  Walthard  (1893),  in  experiments  with  mucus  from  the  cer- 
vix uteri,  was  not  able  to  demonstrate  any  germicidal  action,  but 
arrived  at  the  conclusion  that  it  prevents  the  development  of  bacteria 
simply  because  it  is  an  unfavorable  medium.  Various  bacteria  were 
planted  upon  the  surface  of  cervical  mucus  in  Petri  dishes,  and  placed 
in  the  incubating  oven,  but  all  failed  to  grow. 

Nucleins  from  animal  and  vegetable  cells  have  been  shown  by 
Professor  Vaughan  and  his  associates  (1893)  to  possess  considerable 
germicidal  power.  The  nucleins  of  animal  origin  were  obtained  from 
the  testes  of  dogs  and  rats.  Dissolved  in  a  0.5-per-cent  solution  of 


ACTION   OF   BLOOD    SEBUM  AND  'OTHEK   OKGANIC   LIQUIDS.      211 

caustic  potash  and  then  diluted  with  four  volumes  of  physiologic  salt 
solution  the  germicidal  activity  was  shown  by  the  facts  that  Staphylo- 
coccus  pyogenes  aureus,  and  the  anthrax  bacillus  without  spores, 
failed  to  grow  after  twenty  minutes'  exposure.  Kossel  (1893)  has 
obtained  similar  results  with  nucleins  from  the  thymus  gland  of  the 
calf. 

Urine. — The  experiments  of  Lehmann  show  that  fresh  urine  has 
a  decided  germicidal  power  for  the  cholera  spirillum  and  the  anthrax 
bacillus,  and  no  doubt  for  other  bacteria  as  well.  To  what  constitu- 
ent of  the  urine  this  is  due  has  not  been  determined,  but  it  may  be 
due  to  the  uric  acid  present. 


XIII. 

PRACTICAL  DIRECTIONS  FOR  DISINFECTION. 

THE  Committee  on  Disinfectants  of  the  American  Public  Health 
Association  (appointed  in  1884),  after  an  extended  investigation  with 
reference  to  the  germicidal  value  of  various  agents,  in  a  final  report 
submitted  in  1887  submits  the  following  "  Conclusions": 

The  experimental  evidence  recorded  in  this  report  seems  to  justify  the 
following  conclusions : 

The  most  useful  agents  for  the  destruction  of  spore-containing  infectious 
material  are — 

1.  Fire.     Complete  destruction  by  burning. 

2.  Steam  underpressure.     105°  C.  (221°  F.)  for  ten  minutes. 

3.  Soiling  in  water  for  half  an  hour. 

4.  Chloride  of  lime.1    A  four-per-cent  solution. 

5.  Mercuric  chloride.     A  solution  of  1 :  500. 

For  the  destruction  of  infectious  material  which  owes  its  infecting  power 
to  the  presence  of  microorganisms  not  containing  spores,  the  committee  rec- 
ommends— 

1.  Fire.     Complete  destruction  by  burning. 

2.  Boiling  in  water  for  ten  minutes. 

3.  Dry  heat.     110°  C.  (230°  F  )  for  two  hours. 

4.  Chloride  of  lime.     A  two-per-cent  solution. 

5.  Solution  of  chlorinated  soda.*    A  ten-per-cent  solution. 

6.  Mercuric  chloride.     A  solution  of  1 :  2,000. 

7.  Carbolic  acid.     A  nve-per-cent  solution. 

8.  Sulphate  of  copper.    A  ft ve-per-ceiit  solution. 

9.  Chloride  of  zinc.    A  ten-per-cent  solution. 

10.  Sulphur  dioxide.*  Exposure  for  twelve  hours  to  an  atmosphere  con- 
taining at  least  four  volumes  per  cent  of  this  gas  in  presence  of 
moisture. 

The  committee  would  make  the  following  recommendations  with  refe- 
rence to  the  practical  application  of  these  agents  for  disinfecting  purposes : 

FOR  EXCRETA. 

(a)  In  the  sick-room : 

1.  Chloride  of  lime  in  solution,  four  per  cent. 
In  the  absence  of  spores : 

2.  Carbolic  acid  in  solution,  five  per  cent. 

3.  Sulphate  of  copper  in  solution,  five  per  cent. 

1  Should  contain  at  least  twenty-five  per  cent  of  available  chlorine. 
8  Should  contain  at  least  three  per  cent  of  available  chlorine. 
3  This  will  require  the  combustion  of  between  three  and  four  pounds  of  sulphur 
for  every  thousand  cubic  feet  of  air  space. 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION. 

(b)  In  privy  vaults: 

1.  Mercuric  chloride  in  solution,  1:  500. l 

2.  Carbolic  acid  in  solution,  five  per  cent. 

(c)  For  the  disinfection  and  deodorization  of  the  surface  of  masses  of  or- 
ganic material  in  privy  vaults,  etc. : 

Chloride  of  lime  in  powder. 

FOR  CLOTHING,    BEDDING,    ETC. 

(a)  Soiled  underclothing1,  bed  linen,  etc. : 

1.  Destruction  by  fire,  if  of  little  value. 

2.  Boiling  for  at  least  half  an  hour. 

3.  Immersion  in  a  solution  of  mercuric  chloride  of  the  strength  of 

1 : 2,000  for  four  hours. 

4.  Immersion  in  a  two-per-cent  solution  of  carbolic  acid  for  four  hours. 
(6)  Outer  garments  of  wool  or  silk,  and  similar  articles,  which  would  be 

injured  by  immersion  in  boiling  water  or  in  a  disinfecting  solution: 

1.  Exposure  in  a  suitable  apparatus  to  a  current  of  steam  for  ten  min- 

utes. 

2.  Exposure  to  dry  heat  at  a  temperature  of  110°  C.  (230°  F.)  for  two 

hours. 
(3)  Mattresses  and  blankets  soiled  by  the  discharges  of  the  sick: 

1.  Destruction  by  fire. 

2.  Exposure  to  superheated  steam,  105°  C.  (221°  F.),  for  ten  minutes. 

(Mattresses  to  have  the  cover  removed  or  freely  opened.) 

3.  Immersion  in  boiling  water  for  half  an  hour. 

FURNITURE  AND   ARTICLES   OF  WOOD,    LEATHER,    AND   PORCELAIN. 

Washing,  several  times  repeated,  with— 
1.  Solution  of  carbolic  acid,  two  per  cent. 

FOR  THE    PERSON. 

The  hands  and  general  surface  of  the  body  of  attendants  of  the  sick,  and 
of  convalescents,  should  be  washed  with — 

1.  Solution  of  chlorinated  soda  diluted  with  nine  parts  of  water,  1 : 10. 

2.  Carbolic  acid,  two-per-cent  solution. 

3.  Mercuric  chloride,  1 : 1,000. 

FOR  THE  DEAD. 

Envelop  the  body  in  a  sheet  thoroughly  saturated  with — 

1.  Chloride  of  lime  in  solution,  four  per  cent. 

2.  Mercuric  chloride  in  solution,  1 :  500. 

3.  Carbolic  acid  in  solution,  five  per  cent. 

FOR  THE   SICK-ROOM   AND   HOSPITAL   WARDS. 

(a)  While  occupied,  wa«h  all  surfaces  with — 

1.  Mercuric  chloride  in  solution,  1: 1,000. 

2.  Carbolic  acid  in  solution,  two  per  cent. 

(b)  When  vacated,  fumigate  with  sulphur  dioxide  for  twelve  hours,  burn- 
ing at  least  three  pounds  of  sulphur  for  every  thousand  cubic  feet  of  air 
space  in  the  room ;  then  wash  all  surfaces  with  one  of  the  above-mentioned 
disinfecting  solutions,  and  afterward  with  soap  and  hot  water;  finally  throw 
open  doors  and  windows,  and  ventilate  freely. 

1  The  addition  of  an  equal  quantity  of  potassium  permanganate  as  a  deodorant, 
and  to  give  color  to  the  solution,  is  to  be  recommended.  [The  writer  no  longer  in- 
dorses this  recommendation.  See  his  paper  on  "  The  Disinfection  of  Excreta,"  ap- 
pended.] 


214  PRACTICAL   DIRECTIONS   FOR   DISINFECTION. 

FOR  MERCHANDISE  AND  THE   MAILS. 

The  disinfection  of  merchandise  and  of  the  mails  will  only  be  required 
under  exceptional  circumstances;  free  aeration  will  usually  be  sufficient.  If 
disinfection  seems  necessary,  fumigation  with  sulphur  dioxide  will  be  the 
only  practicable  method  of  accomplishing-  it  without  injury. 

RAGS. 

(a)  Rag-s  which  have  been  usea  for  wiping  away  infectious  discharges 
should  at  once  be  burned. 

(b)  Rags  collected  for  the  paper-makers  during1  the  prevalence  of  an  epi- 
demic should  be  disinfected,  before  they  are  compressed  in  bales,  by — 

1.  Exposure  to  superheated  steam  of  105°  C.  (221°  F.)  for  ten  minutes. 

2.  Immersion  in  boiling  water  for  half  an  hour. 

SHIPS. 

(a)  Infected  ships  at  sea  should  be  washed  in  every  accessible  place,  and 
especially  the  localities  occupied  by  the  sick,  with — 

1.  Solution  of  mercuric  chloride,  1 : 1,000. 

2.  Solution  of  carbolic  acid,  two  per  cent. 

The  bilge  should  be  disinfected  by  the  liberal  use  of  a  strong  solution  of 
mercuric  chloride. 

(b)  Upon  arrival  at  a  quarantine  station,  an   infected  ship   should  at 
once  be  fumigated  with  sulphurous  acid  gas,  using  three  pounds  of  sulphur 
for  every  thousand  cubic  feet  of  air  space ;  the  cargo  should  then  be  dis- 
charged on  lighters;  a  liberal  supply  of  the  concentrated  solution  of  mercuric 
chloride  (four  ounces  to  the  gallon)  should  be  thrown  into  the  bilge,  and  at 
the  end  of  twenty- four  hours  the  bilge  watei  should  be  pumped  out  and  re- 
placed with  pure  sea  water ;  this  should  be  repeated.    A  second  fumigation, 
after  the  removal  of  the  cargo,  is  recommended;  all  accessible  surf  aces  should 
be  washed  with  one  of  the  disinfecting  solutions  heretofore  recommended, 
and  subsequently  with  soap  and  hot  water. 

FOR  RAILWAY  CARS. 

The  directions  given  for  the  disinfection  of  dwellings,  hospital  wards,  and 
ships  apply  as  well  to  infected  railway  cars.  The  treatment  of  excreta  with 
ja  disinfectant,  before  they  are  scattered  along  the  tracks,  seems  desirable  at 
all  times  in  view  of  the  fact  that  they  may  contain  infectious  germs.  Dur- 
ing the  prevalence  of  an  epidemic  of  cholera  this  is  imperative.  For  this 
purpose  the  standard  solution  of  chloride  of  lime  is  recommended. 

DISINFECTION  BY   STEAM. 

The  Committee  on  Disinfectants,  in  tne  above-quoted  "Conclu- 
sions," recommends  the  use  of  "  steam  under  pressure,  105°  C.  (221° 
F.),  for  ten  minutes"  for  the  destruction  of  spore-containing  infec- 
tious material.  The  spores  of  all  known  pathogenic  bacteria  are  de- 
stroyed by  a  temperature  of  100°  C.  maintained  for  five  minutes,  and 
in  view  of  this  fact  the  temperature  fixed  by  the  committee  is  ample, 
and  to  exact  a  higher  temperature  or  longer  exposure  would  be  un- 
reasonable. But  in  practical  disinfection  the  temperature  required 
to  destroy  infectious  material  is  not  the  only  question  to  be  considered. 
Economy  in  the  construction  and  operation  of  the  steam  disinfecting 
apparatus  must  have  due  attention,  and  an  important  point  relates 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION.  215 

to  the  penetration  of  porous,  non-conducting  articles,  such  as  rolls  of 
blankets,  clothing,  etc.  These  points  have  been  the  subject  of  nu- 
merous experimental  investigations,  and  the  principles  involved 
have  been  elucidated,  especially  by  the  investigations  of  Esmarch 
(1887),  of  Budde  (1889),  and  of  Teuschner  (1890). 

It  has  been  shown  that  streaming  steam  is  more  effective  than 
confined  steam  at  the  samq  temperature,  because  it  penetrates  porous 
objects  more  quickly.  Also  that  superheated,  "  dry  "  steam  is  not  as 
effective  as  flowing  steam  at  100°  C. ;  on  the  other  hand,  it  corre- 
sponds in  effectiveness  with  dry  air,  and  the  temperature  must  be 
raised  to  1-40°  to  150°  C.  in  order  to  quickly  destroy  the  spores  of 
bacilli. 

Esmarch's  investigations  show  that  streaming  steam  penetrates 
porous  objects,  like  rolled  blankets,  more  readily  than  confined 
steam ;  but  the  later  researches  of  Budde  and  of  Teuschner  show 
that  a  temperature  of  100°  C.  is  more  rapidly  reached  in  the  interior 
of  such  rolls  when  the  flowing  steam  is  under  pressure.  With  the 
same  pressure  (fifteen  pounds)  a  temperature  of  100°  C.  was  reached 
in  two  and  one-half  minutes  when  the  steam  was  flowing,  and  in 
eleven  minutes  by  steam  at  rest  (Budde).  Intermittent  pressure 
was  not  found  by  Budde  to  present  any  advantages  over  continuously 
flowing  steam  ;  on  the  contrary,  the  time  of  penetration  was  longer. 

Teuschner,  whose  investigations  are  the  most  recent,  arrives  at 
the  following  conclusions  : 

1.  Strongly  superheated  steam  is  not  to  be  recommended  for  practical 
disinfection.     On  the  contrary,  a  slight  superheating-  of  the  steam,  such  as 
occurs  in  the  apparatus  of  Schimmel,  is  not  objectionable. 

2.  Those  forms  of  apparatus  in  which  the  steam  enters  from  above  are 
much  safer  and  quicker  in  their  disinfecting  action  than  those  in  which  this 
is  not  the  case.     In  the  construction  of  such  apparatus  care  must  be  taken, 
in  order  to  secure  penetration  of  the  objects,  that  the  air  and  steam  have  a 
free  escape  below. 

3..  Disinfection  is  hastened  by  previously  warming  the  apparatus. 

4.  The  most  rapid  disinfecting  action  is  secured  by  the  use  of  streaming 
steam  in  a  state  of  tension  (under  pressure). 

5.  Objects  which  have  been  .in  contact  with  fatty  or    oily  substances 
require  a  longer  time  for  disinfection  than  those  which  have  not. 

6.  To  accomplish  disinfection  it  is  necessary  to  expel,  as  completely  as 
possible,  all  air  from  the  objects  to  be  disinfected,  and  also  to  secure  a  suffi- 
cient condensation  of  the  steam. 

7.  The  condensation  of  the  steam  advances  in  a  sharply  defined  line 
from  the  periphery  to  the  centre  of  porous  objects. 

8.  The  temperature  necessary  for  disinfection  is  only  found  in  the  zone 
where  condensation  has  already  taken  place. 

9.  Only  a  few  centimetres  from  the  zone  in  which  the  temperature  is 
100°  C. — when  disinfection  is  incomplete— there  may  be  places  in  which 
the  temperature  is  40°  C,  or  more  below  the  boiling  point. 


216  PRACTICAL   DIRECTIONS   FOR  DISINFECTION. 


DISINFECTION   BY   FORMALDEHYDE   GAS. 

Recent  experiments  have  demonstrated  the  valuable  germicidal 
properties  of  formaldehyde  gas.  Owing  to  its  superior  germicidal 
value  and  non-toxic  properties,  it  has  to  a  considerable  extent  taken 
the  place  of  sulphur  dioxide  as  a  gaseous  disinfectant.  In  making 
practical  use  of  this  agent  a  suitable  apparatus  will  be  required.  For 
the  disinfection  of  a  room  with  its  contents,  freely  exposed  for  sur- 
face disinfection,  one  pound  of  formalin  should  be  volatilized  for 
each  thousand  cubic  feet  of  air  space — the  time  of  exposure  to  the 
disinfecting  action  of  the  gas  being  not  less  than  twelve  hours. 
When  paraform  is  used  the  amount  required  will  be  sixty  grammes 
to  one  thousand  cubic  feet  (Novy).  In  the  absence  of  any  appa- 
ratus satisfactory  results  have  been  obtained  by  the  Department  of 
Health  of  the  city  of  Chicago,  as  follows: 

"Ordinary  bed  sheets  were  employed  to  secure  an  adequate  eyaporatory 
surface,  and  these,  suspended  in  the  room,  were  simply  sprayed  with  a  forty- 
per-cent  solution  of  formalin  through  a  common  watering-pot  rose-head.  A 
sheet  of  the  usual  size  and  quality  will  carry  from  one  hundred  and  fifty  to 
one  hundred  and  eighty  cubic  centimetres  of  the  solution  without  dripping, 
and  this  quaatity  has  been  found  sufficient  for  the  disinfection  of  one  thousand 
cubic  feet  of  space.  Of  course,  the  sheets  may  be  modified  to  any  necessary 
number.  .  .  .  Surface  disinfection  was  thorough,  while  a  much  greater  de- 
gree of  penetration  was  shown  than  that  secured  by  any  other  method." 

Formalin  may  also  be  used  in  the  disinfection  of  rooms  and  their 
contents  by  spraying  all  exposed  surfaces. 

Experiments  made  by  Kinyoun  and  others  show  that  formalde- 
hyde gas  does  not  injure  the  color  or  textile  strength  of  fabrics  of 
wool,  silk,  cotton,  or  linen,  and  that  it  has  no  injurious  action  upon 
furs,  leather,  copper,  brass,  nickel,  zinc,  polished  steel  or  gilt  work. 
Iron  and  unpolished  steel  are  attacked  by  the  gas. 

DISINFECTION   OF   THE   HANDS. 

The  importance  of  a  reliable  method  of  disinfecting  the  hands  of 
surgeons,  obstetricians,  and  nurses  after  they  have  been  in  contact 
with  infectious  material  from  wounds,  puerperal  discharges,  etc. ,  is 
now  fully  recognized,  and  some  surgeons  consider  it  necessary  to 
completely  sterilize  the  hands  before  undertaking  any  surgical  opera- 
tion which  will  bring  them  in  contact  with  the  freshly-cut  tissues. 
The  numerous  experiments  which  have  been  made  with  a  view  to 
ascertaining  the  best  method  of  accomplishing  such  sterilization  of 
the  hands  show  that  it  is  by  no  means  a  simple  matter  to  effect  it, 
and  especially  to  insure  the  destruction  of  microorganisms  con- 
cealed beneath  the  finger  nails.  Furbringer,  in  an  extended  series 
of  experiments  (1888),  found  that  a  preliminary  cleansing  with  soap 


PRACTICAL   DIRECTIONS   FOR   DISINFECTION.  217 

and  a  brush  was  even  more  important  than  the  degree  of  potency  of 
the  disinfecting  wash  subsequently  applied.  He  recommends  the 
following  procedure  : 

1.  Remove  all  visible  dirt  from  beneath  and  around  the  nails. 

2.  Brush  the  spaces  beneath  the  nails  with  soap  and  hot  water 
for  a  minute. 

3.  Wash  for  a  minute  in  alcohol  (not  below  eighty  per  cent),  and, 
before  this  evaporates,  in  the  following  solution  : 

4.  Wash  thoroughly  for  a  minute  in  a  solution  containing  1  :  500 
of  mercuric  chloride  or  three  per  cent  of  carbolic  acid. 

Roux  and  Reynes  tested  the  above  method  of  Fiirbringer,  and 
found  that  it  gave  better  results  than  others  previously  proposed,  al- 
though not  always  entirely  successful  in  securing  complete  steriliza- 
tion. 

Boll  has  recently  (1890)  reported  favorable  results  from  the  fol- 
lowing method  : 

1.  Cleanse  the  finger  nails  from,  visible  dirt  with  knife  or  nail  scissors. 

2.  Brush  the  hands  for  three  minutes  with  hot  water  and  potash  soap. 

3.  Wash  for  half  a  minute  in  a  three-per-cent  solution  of  carbolic  acid, 
and  subsequently  in  a  1  :  2,000  solution  of  mercuric  chloride. 

4.  Rub  the  spaces  beneath  the  nails  and  around  their  margins  with  iodo- 
form  gauze  wet  in  a  five-per-ceiit  solution  of  carbolic  acid. 

Welch,  as  a  result  of  extended  experiments  made  at  the  Johns 
Hopkins  Hospital,  recommends  the  following  procedure  : 

1.  The  nails  are  kept  short  and  clean. 

2.  The  hands  are  washed  thoroughly  for  several  minutes  with  soap  and 
water,  the  water  being  as  warm  as  can  be  comfortably  borne,  and  being  fre- 
quently changed.     A  brush  sterilized  by  steam  is  used.     The  excess  of  soap 
is  washed  off  with  water. 

3.  The  hands  are  immersed  for  one  or  two  minutes  in  a  warm  saturated 
solution  of  permanganate  of  Dotash  and  are  rubbed  over  thoroughly  with  a 
sterilized  swab. 

4.  They  are   then  placed  in  a  warm  saturated  solution  of  oxalic  acid, 
where  they  remain  until  complete   decolorization  of  the    permanganate 
occurs. 

5.  They  are  then  washed  off  with  sterilized  salt  solution  or  water. 

6.  They  are  immersed  for  two  minutes  in  sublimate  solution,  1  :  500. 
The  bacteriological  examination  of  the  skin  thus  treated  yields  almost 

uniformly  negative  results,  the  material  for  the  cultures  being  taken  from 
underneath  and  around  the  nails.  This  is  the  procedure  now  employed  in 
the  gynecological  and  surgical  wards  of  the  hospital. 

THE   DISINFECTION   OF   EXCRETA. 

The  contents  of  privy  vaults  and  cesspools  should  never  be  allowed 
to  accumulate  unduly  or  to  become  offensive.  By  frequent  removal, 
and  by  the  liberal  use  of  antiseptics,  such  necessary  receptacles  of 
filth  should  be  kept  in  a  sanitary  condition.  The  absorbent  deodo- 


218  PRACTICAL   DIRECTIONS   FOR   DISINFECTION. 

rants,  such  as  dry  earth  or  pounded  charcoal,  or  the  chemical  de- 
odorants and  antiseptics,  such  as  chloride  of  zinc,  sulphate  of  iron, 
etc.,  will,  under  ordinar}7  circumstances,  prevent  such  places  from 
becoming  offensive.  Disinfection  will  be  required  only  when  it  is 
known  or  suspected  that  infectious  material,  such  as  the  dejections 
of  patients  with  cholora,  yellow  fever,  or  typhoid  fever,  has  been 
thrown  into  the  receptacles. 

In  the  Manual  for  the  Medical  Department  of  the  United  States 
Army  the  following  directions  are  given : 

92.  When  accumulations  of  organic  material  undergoing-  decomposition 
cannot  be  removed  or  buried,  they  may  be  treated  with  an  antiseptic  solu- 
tion, or  with  freshly  burned  quicklime.     Quicklime  is  also  a  valuable  disin- 
fectant, and  may  be  substituted  for  the  more  expensive  chloride  of  lime  for 
disinfection  of  typhoid  and  cholera  excreta,  etc.     For  this  purpose  freshly 
prepared  milk  of  lime  should  be  used,  containing  about  one  part,  by  weight, 
of  hydrate  of  lime,  to  eight  of  water. 

93.  During  the  prevalence  of  an  epidemic,  or  when  there  is  reason  to 
believe  that  infectious  material  has  been  introduced  from  any  source,  latrines 
and  cesspools  may  be  treated  with  milk  of  lime,  in  the  proportion  of  five 
parts  to  one  hundred  parts  of  the  contents  of  the  vault,  and  the  daily  addi- 
tion of  ten  parts  for  one  hundred  parts  of  daily  increment  of  faeces. 

According  to  Behring,  lime  has  about  the  same  germicidal  value 
as  the  other  caustic  alkalies,  and  destroys  the  cholera  spirillum  and 
the  bacillus  of  typhoid  fever,  of  diphtheria,  and  of  glanders  after 
several  hours'  exposure,  in  the  proportion  of  fifty  cubic  centimetres 
normal-lauge  per  litre.  Wood  ashes  or  lye  of  the  same  alkaline 
strength  may  therefore  be  substituted  for  quicklime. 

Finally,  it  must  not  be  forgotten  that  we  have  a  ready  means  of 
disinfecting  excreta  in  the  sick-room  or  its  vicinity  by  the  application 
of  heat.  Exact  experiments,  made  by  the  writer  and  others,  show 
that  the  thermal  death-point  of  the  following  pathogenic  bacteria, 
and  of  the  kinds  of  virus  mentioned,  is  below  60°  C.  (140°  F.) : 
Spirillum  of  cholera,  bacillus  of  anthrax,  bacillus  of  typhoid  fever, 
bacillus  of  diphtheria,  bacillus  of  glanders,  diplococcus  of  pneu- 
monia (Micrococcus  Pasteuri),  streptococcus  of  erysipelas,  staphylo- 
cocci  of  pus,  micrococcus  of  gonorrhoea,  vaccine  virus,  sheep-pox 
virus,  hydrophobia  virus.  Ten  minutes'  exposure  to  the  tempera- 
ture mentioned  may  be  relied  upon  for  the  disinfection  of  material 
containing  any  of  these  pathogenic  organisms,  except  the  anthrax 
bacillus  when  in  the  stage  of  spore  formation.  The  use,  therefore, 
of  boiling  water  in  the  proportion  of  three  or  four  parts  to  one 
part  of  the  material  to  be  disinfected  may  be  safely  recommended 
for  such  material.  Or,  better  still,  a  ten-per-cent  solution  of  sulphate 
of  iron  or  of  chloride  of  zinc  at  the  boiling-point  may  be  used  in  the 
same  way  (three  parts  to  one). 


PART  THIRD. 


PATHOGENIC  BACTERIA. 

I.  MODES  OF  ACTION.    II.   CHANNELS  OF  INFECTION.    III.   SUSCEPTIBILITY  AND 

IMMUNITY.     IV.  PROTECTIVE  INOCULATIONS.      V.   PYOGENIC  BACTERIA.    VI. 

BACTERIA  IN  CROUPOUS  PNEUMONIA.   VII.  PATHOGENIC  MICROCOCCI  NOT 

DESCRIBED    IN    SECTIONS   V.    AND    VI.       VIII.     TlIE    BACILLUS    OF   AN- 
THRAX.     IX.    THE    BACILLUS    OF    TYPHOID    FEVER.     X.   BAC- 
TERIA IN  DIPHTHERIA.    XI.    BACILLUS  OF  INFLUENZA.    XII. 
BACILLI  IN  CHRONIC  INFECTIOUS  DISEASES.   XIII.  BACILLI 

WHICH    PRODUCE    SEPTICAEMIA    IN    SUSCEPTIBLE   ANI- 
MALS.   XIV.  PATHOGENIC  AEROBIC  BACILLI  NOT 

DESCRIBED     IN     PREVIOUS      SECTIONS.       XV. 

BACTERIA    OF    PLANT  DISEASES.    XVI. 

PATHOGENIC     ANAEROBIC  BACILLI. 

XVII.     PATHOGENIC  SPIRILLA. 


PAET    THIED. 
PATHOGENIC    BACTERIA. 


I. 

MODES  OF  ACTION. 

MANY  of  the  saprophytic  bacteria  are  pathogenic  for  man,  or  for 
one  or  more  species  of  the  lower  animals,  when  by  accident  or  ex- 
perimental inoculation  they  obtain  access  to  the  body  ;  these  may  be 
designated  facultative  parasites.  Other  species  which,  for  a  time 
at  least,  are  able  to  lead  a  saprophytic  mode  of  life  have  their  nor- 
mal habitat  in  the  bodies  of  infected  animals,  in  which  they  produce 
specific  infectious  diseases.  To  this  class  belong  the  cholera  spirillum, 
the  anthrax  bacillus,  the  bacillus  of  typhoid  fever,  and  various  other 
microorganisms  which  are  the  cause  of  specific  infectious  diseases  in 
some  of  the  lower  animals.  These  we  may  speak  of  as  parasites 
and  facultative  saprophytes.  Still  others  are  strict  parasites  and 
do  not  find  the  conditions  for  their  development  outside  of  the  bodies 
of  the  animals  which  they  infest,  except  under  the  special  conditions 
in  which  bacteriologists  have  succeeded  in  cultivating  some  of  them. 
The  best  known  strict  parasites  are  the  tubercle  bacillus,  the  bacillus 
of  leprosy,  the  spirillum  of  relapsing  fever,  and  the  micrococcus  of 
gonorrhoea. 

There  can  be  but  little  doubt  that  even  the  strict  parasites,  at  some 
time  in  the  past,  were  also  saprophytes,  and  that  the  adaptation  to  a 
parasitic  mode  of  life  was  gradually  effected  under  the  laws  of  natural 
selection.  In  a  previous  chapter  (Section  III.,  Part  Second)  we  have 
referred  to  the  modifications  in  biological  characters  which  may 
occur  as  a  result  of  special  conditions  of  environment.  Thus  we  may 
obtain  non-chromogenic  varieties  of  species  which  usually  produce 
pigment,  or  non-pathogenic  varieties  of  bacteria  which  are  usually 
pathogenic.  There  is  also  evidence  that  the  tubercle  bacillus,  a  strict 


222  MODES   OF   ACTION. 

parasite,  may  be  so  modified,  by  cultivation  for  successive  genera- 
tions in  a  culture  medium  containing  glycerin,  that  it  will  finally 
grow  in  ordinary  beef  infusion,  thus  showing  a  tendency  to  adapt 
itself  to  a  saprophytic  mode  of  life. 

Some  of  the  saprophytic  bacteria  are  indirectly  pathogenic  by 
reason  of  their  power  to  multiply  in  articles  of  food,  such  as  milk, 
cheese,  fish,  sausage,  etc.,  and  there  produce  poisonous  ptomaines 
which,  when  these  articles  are  ingested,  give  rise  to  various  morbid 
symptoms,  such  as  vomiting,  gastric  and  intestinal  irritation,  fever, 
etc.  Or  similar  symptoms  may  result  from  the  multiplication  of 
bacteria  producing  toxic  ptomaines  in  the  alimentary  canal.  No 
doubt  gastric  and  intestinal  disorders  are  largely  due  to  this  cause, 
and  may  be  induced  by  a  variety  of  saprophytic  bacteria  when  these 
establish  themselves  in  undue  numbers  in  any  portion  of  the  ali- 
mentary tract.  In  Asiatic  cholera  the  same  thing  occurs,  but  with 
more  fatal  results  from  the  introduction  of  the  East  Indian  cholera 
germ  discovered  by  Koch.  This  is  pathogenic  for  man,  because  it  is 
able  to  multiply  rapidly  in  the  human  intestine,  and  there  produces  a 
toxic  substance  which,  being  absorbed,  gives  rise  to  the  morbid  pheno- 
mena of  the  disease.  The  spirillum  itself  does  not  enter  the  blood  or 
invade  the  tissues,  except  to  a  limited  extent  in  the  mucous  coat  of 
the  intestine,  and  the  true  explanation  of  its  pathogenic  power  is  no 
doubt  that  which  has  been  given. 

Other  microorganisms  invade  the  tissues  and  multiply  in  cer- 
tain favorable  localities,  but  have  not  the  power  of  developing  in  the 
blood,  in  which  they  are  only  found  occasionally  and  in  very  small 
numbers  or  not  at  all.  Thus  the  typhoid  bacillus  locates  itself  in  the 
intestinal  glands,  in  the  spleen,  and  in  the  liver,  forming  colonies  of 
limited  extent,  and  evidently  not  finding  the  conditions  extremely 
favorable  for  its  growth,  inasmuch  as  it  does  not  take  complete  pos- 
session of  these  organs.  The  symptoms  which  result  from  its  pre- 
sence are  doubtless  partly  due  to  local  irritation,  disturbance  of  func- 
tion, and,  in  the  case  of  the  intestinal  glands,  necrotic  changes 
induced  by  it.  But  in  addition  to  this  its  pathogenic  action  depends 
upon  the  production  of  a  poisonous  ptomaine  which  has  been  isolated 
and  studied  by  the  German  chemist  Brieger  (typhotoxin). 

Certain  saprophytic  bacteria,  when  injected  beneath  the  skin  of  a 
susceptible  animal,  multiply  at  the  point  of  inoculation  and  invade 
the  surrounding  tissues,  giving  rise  in  some  instances  to  the  forma- 
tion of  a  local  abscess,  in  others  to  an  infiltration  of  the  tissues  with 
bloody  serum,  and  in  others  to  extensive  necrotic  changes.  These 
local  changes  are  due  not  simply  to  the  mechanical  presence  of  the 
microorganisms  which  induce  them,  but  to  chemical  products  evolved 
during  the  growth  of  these  pathogenic  bacteria.  Indeed,  their  patho- 


MODES   OF   ACTION.  223 

genie  power  evidently  depends,  in  some  instances  at  least,  upon  these 
toxic  products  of  their  growth,  by  which  the  vital  resisting  power  of 
the  tissues  is  overcome. 

Among  the  bacteria  which  in  this  way  produce  extensive  local 
inflammatory  and  necrotic  changes  are  certain  anaerobic  species 
found  in  the  soil  and  in  putrefying  material,  such  as  the  bacillus  of 
malignant  oedema  and  the  writer's  Bacillus  cadaveris.  The  bacillus 
of  symptomatic  anthrax,  an  infectious  disease  of  cattle,  acts  in  the 
same  way.  All  of  these  produce  toxic  substances  which  have  a  very 
pronounced  local  action  upon  the  tissues  invaded  by  them.  Other 
bacteria,  while  they  develop  chiefly  in  the  vicinity  of  the  point  of 
entrance — by  accident  or  by  inoculation — produce  a  potent  toxic  sub- 
stance which  gives  rise  to  general  symptoms  of  a  serious  character, 
such  as  tetanic  convulsions  (bacillus  of  tetanus)  or  intense  fever  and 
nervous  phenomena  (micrococcus  of  erysipelas).  Again,  the  local 
irritation  resulting  from  the  presence  of  parasitic  bacteria  may  pri- 
marily give  rise  to  the  formation  of  new  growths  having  a  low  grade 
of  vitality,  which  later  may  undergo  necrotic  changes,  as  in  tubercu- 
losis, glanders,  and  leprosy.  In  this  case  constitutional  symptoms 
are  not  present,  or  are  of  a  mild  character  during  the  development 
of  these  new  formations,  which  apparently  result  from  the  local  ac- 
tion of  substances  eliminated  during  the  growth  of  the  parasite, 
rather  than  from  its  simple  presence.  This  is  an  inference  based 
upon  the  fact  that  non-living  particles,  or  even  living  parasites,  as  in 
trichinosis,  do  not  produce  similar  new  growths  composed  of  cells, 
but  become  encysted  in  a  fibrous  capsule. 

In  pneumonia  we  have  a  local  process  in  which  one  or  more  lobes 
of  the  lung  are  invaded  by  a  pathogenic  micrococcus  (Micrococcus 
pneumonise  crouposoe)  which  induces  a  fibrinous  exudation  that  com- 
pletely fills  the  air  cells.  How  far  the  symptoms  of  the  disease  are 
due  to  the  local  inflammation  and  disturbance  of  function,  and  to 
what  extent  they  may  be  due  to  the  absorption  of  a  soluble  toxic 
substance  evolved  as  a  result  of  the  growth  of  the  micrococcus,  has 
not  been  determined.  But  the  mild  character  of  the  general  symp- 
toms when  a  limited  area  of  lung  tissue  is  involved  leads  to  the  in- 
ference that  the  pathogenic  power  of  this  particular  pathogenic 
microorganism  is  chiefly  exercised  locally. 

The  pus  cocci  and  various  other  saprophytic  bacteria,  when  intro- 
duced beneath  the  skin,  give  rise  to  the  formation  of  abscesses,  un- 
attended by  any  very  considerable  general  disturbance  ;  and  also  to 
secondary  purulent  accumulations — metastatic  abscesses. 

That  this  is  not  due  simply  to  their  mechanical  presence  is  shown 
by  the  fact  that  powdered  glass  and  other  inert  substances,  when 
thoroughly  sterilized,  do  not  give  rise  to  pus  formation  when  intro- 


224  MODES   OF   ACTION. 

duced  beneath  the  skin  or  injected  into  the  cavity  of  the  abdomen. 
On  the  other  hand,  it  has  been  demonstrated  by  the  experiments  of 
Grawitz,  De  Bary,  and  others  that  certain  chemical  substances 
which  act  as  local  irritants  when  brought  in  contact  with  the  tissues 
may  induce  pus  formation  quite  independently  of  microorganisms  : 
nitrate  of  silver,  oil  of  turpentine,  and  strong  liquor  ammonise  have 
been  shown  to  possess  this  power.  And  it  has  been  demonstrated  by 
the  recent  experiments  of  Buchner  that  sterilized  cultures  of  a  long 
list  of  different  bacteria — seventeen  species  tested — give  rise  to  sup- 
puration when  introduced  into  the  subcutaneous  tissues. 

Buchner  has  further  shown  that  this  property  of  inducing  pus  for- 
mation resides  in  the  dead  bacterial  cells  and  not  in  soluble  products 
present  in  the  cultures.  For  the  clear  fluid  obtained  by  passing 
these  sterilized  cultures  through  a  porcelain  filter  gave  a  negative  re- 
sult, while  the  bacteria  retained  by  the  filter,  although  no  longer 
capable  of  development,  having  been  killed  by  heat,  invariably 
caused  suppuration. 

Individuals  suffering  from  malnutrition  are  more  susceptible  to 
invasion  by  specific  disease  germs  or  by  the  common  pus  cocci 
than  are  those  in  vigorous  health.  Thus  the  sufferers  from  starva- 
tion, from  crowd  poisoning,  sewer-gas  poisoning,  etc.,  are  not  only 
liable  to  be  early  victims  during  the  prevalence  of  an  epidemic  dis- 
ease, but  are  very  subject  to  abscesses,  boils,  ulcers,  etc.  A  slight 
abrasion  in  such  an  individual,  inoculated  by  the  ever-present  pus 
cocci,  may  give  rise  to  an  obstinate  ulcer  or  a  phlegmonous  inflam- 
mation. 

In  the  same  way  some  of  the  ordinary  saprophytes,  which  usually 
have  no  pathogenic  power,  may  be  pathogenic  for  an  animal  whose 
strength  is  reduced  by  disease  or  injury.  Thus  necrotic  changes 
may  occur  in  injured  tissues,  or  in  those  which  have  a  deficient  blood 
supply — from  occlusion  of  an  artery,  for  example — due  to  the  presence 
of  putrefactive  bacteria  which  are  incapable  of  development  in  the 
circulation  of  a  healthy  animal  or  in  healthy  tissues.  We  may  also 
have  a  progressive  gangrene,  due  to  infection  of  wounds  by  bacteria 
which  are  able  to  invade  healthy  tissues.  This  is  seen  in  the  so- 
called  hospital  gangrene,  which  is  undoubtedly  due  to  microorgan- 
isms, although  the  species  concerned  in  its  production  has  not  been 
determined,  owing  to  the  fact  that  modern  bacteriologists  have  had 
few,  if  any,  opportunities  for  studying  it.  The  history  of  the  disease, 
its  rapid  extension  in  infected  surgical  wards,  the  extensive  slough- 
ing which  occurs  within  a  few  hours  in  previously  healthy  wounds, 
and  the  effect  of  deep  cauterization  by  the  hot  iron,  nitric  acid,  or 
bromine  in  arresting  the  progress  of  the  disease,  all  support  this  view 
of  its  etiology.  Whether  it  is  due  to  a  specific  pathogenic  micro- 


MODES   OF   ACTION.  225 

organism,  or  to  exceptional  pathogenic  power  acquired  by  some  one 
of  the  common  bacteria  which  infest  suppurating  wounds,  cannot  be 
determined  in  the  absence  of  exact  experiments  by  modern  methods. 
But  the  latter  view  has  seemed  to  the  writer  the  most  probably  cor- 
rect. There  are  many  facts  which  go  to  show  that  pathogenic  viru- 
lence may  be  increased  by  cultivation  in  animal  fluids,  and  where 
wounded  men  are  brought  together  under  unfavorable  sanitary  con- 
ditions, as  has  been  the  case  where  hospital  gangrene  has  made  its 
appearance,  it  may  be  that  some  common  saprophyte  acquires  the 
power  of  invading  the  exposed  tissues  instead  of  simply  feeding  upon 
the  secretions  which  bathe  its  surface. 

Koch  has  described  a  progressive  tissue  necrosis  in  mice,  due  to  a 
streptococcus,  which  he  first  obtained  by  inoculating  a  mouse  in  the 
ear  with  putrid  material.  The  morbid  process  is  entirely  local  and 
rapidly  progressive,  causing  a  fatal  termination  in  about  three  days, 
without  invasion  of  the  blood. 

In  diphtheritic  inflammations  of  mucous  membranes  we  have 
a  local  invasion  of  the  tissues  and  a  characteristic  plastic  exudation. 
In  true  diphtheria  the  local  inflammation  and  necrotic  changes  in 
the  invaded  tissues  are  not  sufficient  to  account  for  the  serious  gen- 
eral symptoms,  and  we  now  have  experimental  evidence  that  the 
diphtheria  bacillus  produces  a  very  potent  toxic  substance  to  which 
these  symptoms  are  no  doubt  largely  due.  The  diphtheria  bacillus 
of  Loffler  appears  to  be  the  cause  of  the  fatal  malady  which  goes 
by  this  name,  but  undoubtedly  other  microorganisms  may  be  con- 
cerned in  the  formation  of  diphtheritic  false  membranes.  In  cer- 
tain forms  of  diphtheria,  and  especially  when  it  occurs  as  a  com- 
plication of  scarlet  fever,  measles,  and  other  diseases,  the  Klebs- 
Loffler  bacillus  is  absent,  and  a  streptococcus,  which  appears  to  be 
identical  with  Streptococcus  pyogenes,  is  found  in  considerable  num- 
bers and  is  probably  the  cause  of  the  diphtheritic  inflammation. 
An  epidemic  of  diphtheria  occurring  among  calves  was  studied  by 
Loffler,  and  is  ascribed  by  him  to  his  Bacillus  diphtherise  vitulo- 
rum.  The  same  bacteriologist  has  shown  that  the  diphtheria  of 
chickens  and  of  pigeons  is  due  to  a  specific  bacillus  which  differs 
from  that  found  in  human  diphtheria,  and  which  he  calls  Bacillus 
diphtheria  columbrarum. 

Prof.  Welch  has  studied  the  histological  lesions  produced  by 
filtered  cultures  of  the  diphtheria  bacillus.  Cultures  in  glycerin- 
bouillon,  several  weeks  old,  were  filtered  through  porcelain,  and  the 
sterile  filtrate  was  injected  beneath  the  skin  of  guinea-pigs.  One 
cubic  centimetre  of  this  filtrate  was  injected  into  a  guinea-pig  on 
the  10th  of  December,  and  two  cubic  centimetres  more  on  the 
14th  of  the  same  month.  The  animal  succumbed  at  the  end  of 
15 


226  MODES   OF   ACTION. 

three  weeks  and  five  days  after  the  first  inoculation.  At  the  autopsy 
"  the  lymphatic  glands  of  the  inguinal  and  axillary  regions  were 
found  to  be  enlarged  and  reddened;  the  cervical  glands  were  swollen 
and  the  thyroid  gland  was  greatly  congested.  There  was  a  consider- 
able excess  of  clear  fluid  in  the  peritoneal  cavity.  Both  layers  of  the 
peritoneum  were  reddened,  the  vessels  of  the  visceral  layer  being  es- 
pecially injected.  The  spleen  was  enlarged  to  double  the  average 
size;  it  was  mottled,  and  the  white  follicles  were  distinctly  outlined 
against  the  red  ground.  The  liver  was  dark  in  color  and  contained 
much  blood.  .  .  .  The  kidneys  were  congested  and  the  cut  surface 
was  cloudy.  .  .  .  The  pericardial  sac  was  distended  with  clear  se- 
rum. Under  the  epicardium  were  many  ecchymotic  spots.  The 
lungs  exhibited  areas  of  intense  congestion  or  actual  haemorrhage 
into  the  tissues.  .  .  .  The  histological  lesions  in  this  case  are  identi- 
cal with  those  observed  by  us  in  connection  with  the  inoculation  of 
the  living  organisms." 

To  what  extent  non-specific  catarrhal  inflammations  of  mucous 
membranes  are  caused  by  the  local  action  of  microorganisms  has 
not  been  determined,  but  in  gonorrhoea  the  proof  is  now  considered 
satisfactory  that  the  "  gonococcus  "  of  Neisser  is  the  cause  of  the 
intense  local  inflammation  and  purulent  discharge.  In  this  disease 
the  action  of  the  pathogenic  microorganism  seems  to  be  limited  to 
the  tissues  invaded  by  it,  as  there  is  no  general  systemic  disturbance 
indicating  the  absorption  of  a  toxic  ptomaine. 

Chronic  catarrhal  inflammations  appear,  in  some  cases  at  least, 
to  be  kept  up  by  the  presence  of  microorganisms,  which  are  always 
found  in  the  discharges  from  inflamed  mucous  surfaces. 

The  influence  of  microorganisms,  and  especially  of  the  pus  cocci, 
in  preventing  the  prompt  healing  of  wounds,  is  now  well  established. 
An  extensive  suppurating  wound  or  collection  of  pus,  especially  if 
putrefactive  bacteria  are  present,  causes  fever  and  nervous  symp 
toms,  due  to  the  absorption  of  toxic  products.  More  intense  general 
symptoms  result  from  the  presence  of  the  streptococcus  of  pus  than 
from  the  less  pathogenic  staphylococci ;  this  is  seen  in  erysipelatous 
inflammations  and  in  puerperal  metritis  due  to  the  presence  of  this 
micrococcus.  Like  the  other  pus  cocci,  the  Streptococcus  pyogenes 
does  not  usually  invade  the  blood,  but  when  introduced  into  the  sub- 
cutaneous tissues  it  induces  a  local  inflammatory  process,  with  a  ten- 
dency to  pus  formation,  and  it  invades  the  neighboring  lymph  chan- 
nels, in  which  the  conditions  appear  to  be  especially  favorable  for  its 
multiplication. 

Finally,  certain  pathogenic  bacteria,  when  introduced  into  the 
bodies  of  susceptible  animals,  quickly  invade  the  blood  and  multiply 
in  it.  In  so  doing  they  necessarily  interfere  with  its  physiological 


MODES   OF   ACTION.  227 

functions  by  appropriating  for  their  own  use  material  required  for 
the  nutrition  of  the  tissues ;  and  at  the  same  time  toxic  substances 
are  formed  which  play  an  important  part  in  the  production  of  the 
morbid  phenomena,  which  in  this  class  of  diseases  very  commonly 
lead  to  a  fatal  result.  The  pathogenic  bacteria  which  invade  the 
blood  may  also,  in  certain  cases,  give  rise  to  local  necrosis  and  dis- 
turbance of  function  in  various  organs  in  a  mechanical  way  by 
blocking  up  the  capillaries. 

The  invasion  of  the  blood  which  occurs  in  anthrax  and  in  vari- 
ous forms  of  septicaemia  in  the  lower  animals,  induced  by  subcuta- 
neous inoculation  with  pure  cultures  of  certain  pathogenic  bacteria, 
does  not  generally  immediately  follow  the  inoculation.  Usually  a 
considerable  local  development  first  occurs,  which  gives  rise  to  more 
or  less  inflammation  of  the  invaded  tissues,  and  very  commonly  to 
an  effusion  of  bloody  serum  in  which  the  pathogenic  microorganism 
is  found  in  great  numbers.  Even  in  susceptible  animals  the  blood 
seems  to  offer  a  certain  resistance  to  invasion,  which  is  overcome 
after  a  time  by  the  vast  number  of  the  parasitic  host  located  in  the 
vicinity  of  the  point  of  inoculation,  aided  probably  by  the  toxic  sub- 
stances developed  as  a  result  of  their  vital  activity. 

The  experiments  of  Cheyne  (1886)  seem  to  show  that  in  the  case 
of  very  pathogenic  species,  like  the  anthrax  bacillus  or  Koch's  bacil- 
lus of  mouse  septicaemia,  a  single  bacillus  introduced  subcutaneously 
may  produce  a  fatal  result  in  the  most  susceptible  animals,  while 
greater  numbers  are  required  in  those  which  are  less  susceptible. 
Thus  a  guinea-pig  succumbed  to  general  infection  after  being  inocu- 
lated subcutaneously  with  anthrax  blood  diluted  to  such  an  extent 
that,  by  estimation,  only  one  bacillus  was  present  in  the  fluid  in- 
jected ;  and  a  similar  result  in  mice  was  obtained  with  Bacillus 
murisepticus.  In  the  case  of  the  microbe  of  fowl  cholera  (Bacillus 
septicaemias  haemorrhagicae)  Cheyne  found  that  for  rabbits  the  fatal 
dose  is  300,000  or  more,  that  from  10,000  to  300,000  cause  a  local 
abscess,  and  that  less  than  10,000  produce  no  appreciable  effect. 
The  common  saprophyte  Proteus  vulgaris  was  found  to  be  patho- 
genic for  rabbits  when  injected  into  the  dorsal  muscles  hi  sufficient 
numbers.  But,  according  to  the  estimates  made,  225,000,000  were 
required  to  cause  death,  while  with  doses  of  from  9,000,000  to  112,- 
000,000  a  local  abscess  was  produced,  and  less  than  9,000,000  gave 
an  entirely  negative  result. 

Secondary  infections  occurring  in  the  course  of  specific  infec- 
tious diseases  are  of  common  occurrence.  Thus  a  pneumonia  may 
be  developed  in  the  course  of  an  attack  of  measles  or  of  typhoid 
fever  ;  or  infection  by  the  common  pus  cocci  in  the  course  of  scarlet 
fever,  typhoid  fever,  mumps,  etc.,  may  give  rise  to  local  abscesses, 


228  MODES    OF    ACTION. 

to  endocarditis,  etc.  Again,  mixed  infection  ma}'  be  induced  by 
injecting  simultaneously  into  susceptible  animals  two  species  of  path- 
ogenic bacteria. 

Bumm,  Bockhart,  and  others  have  reported  cases  of  mixed  gonor- 
rhceal  infection  in  which  the  pyogenic  micrococci  gave  rise  to  ab- 
scesses in  the  glands  of  Bartholin,  to  cystitis,  parametritis,  or  to 
"  gonorrhceal  inflammation  "  of  the  knee  joint.  Babes  gives  numer- 
ous examples  of  mixed  infection  in  scarlet  fever  and  in  other  diseases 
of  childhood.  Anton  and  Fiitterer  have  studied  the  question  of 
secondary  infection  in  typhoid  fever.  Karlinski  has  reported  a  case 
of  secondary  infection  with  anthrax  in  a  case  of  typhoid  fever,  infec- 
tion occurring  by  way  of  the  intestine.  Many  other  examples  of 
secondary  or  mixed  infection  are  recorded  in  the  recent  literature  of 
bacteriology  and  clinical  medicine,  but  enough  has  been  said  to  call 
attention  to  the  importance  of  the  subject. 

The  researches  of  Romer,  Kanthack  (1892),  and  others  show  that 
the  injection  of  the  filtered  products  of  certain  bacteria  (Bacillus 
pyocyaneus,  Vibrio  Metchnikovi,  etc.)  produces  a  decided  leucocy- 
tosis  in  the  animals  experimented  upon.  And  a  similar  result,  prob- 
ably from  a  like  cause,  has  been  shown  by  recent  experiments  to 
occur  in  pneumonia  (Billings)  and  other  infectious  diseases. 

Certain  bacterial  products  have  been  shown  by  experiment  to  pro- 
duce fever  when  injected  into  the  circulation  or  beneath  the  skin  of 
lower  animals ;  others  produce  rapid  respiration,  dilatation  of  pupils, 
diarrhoea,  and  paralysis  or  convulsions  (typhotoxin  of  Brieger, 
methyl-guanidin,  etc.) ;  the  toxic  effects  of  some  are  immediate  and 
of  others  more  or  less  remote  (toxalbumin  of  diphtheria) ;  others  have 
a  primary  toxic  effect  which  is  followed  after  a  time  by  toxic  symp- 
toms of  a  different  order  (Pneumobacillus  liquefaciens  bovis). 


II. 

CHANNELS   OF  INFECTION. 

WE  have  abundant  evidence  that  susceptible  animals  may  be  in- 
fected by  the  injection  of  various  pathogenic  bacteria  beneath  the 
skin,  and  accidental  infection  through  an  open  tvound  or  abrasion 
of  the  skin  is  the  common  mode  of  infection  in  tetanus,  erysipelas, 
hospital  gangrene,  and  the  "  traumatic  infectious  diseases"  generally. 
Other  infectious  diseases,  like  anthrax  and  glanders,  are  frequently 
transmitted  in  the  same  way.  We  have  also  satisfactory  evidence 
that  tuberculosis  may  be  transmitted  to  man  by  the  accidental  inocu- 
lation of  an  open  wound  ;  and  in  view  of  the  fact  that  susceptible 
animals  are  readily  infected  in  this  way,  it  would  be  strange  if  it 
were  otherwise. 

The  question  whether  infection  may  occur  through  the  unbroken 
skin  has  been  studied  by  several  bacteriologists  and  an  affirmative 
result  obtained.  Thus  Schimmelbusch  produced  pustules  upon  the 
thigh  in  two  young  persons  suffering  from  pyaemia  by  rubbing  upon 
the  surface  a  pure  culture  of  Staphylococcus  pyogenes  aureus  which 
he  had  obtained  from  the  pus  of  a  furuncle.  The  same  author  also 
succeeded  in  infecting  rabbits  and  guinea-pigs  with  anthrax,  and 
rabbits  with  rabbit  septicaemia,  by  rubbing  pure  cultures  upon 
the  uninjured  skin.  Similar  results  had  previously  been  reported 
by  Roth,  who  also  showed  that  infection  might  occur  through 
the  uninjured  mucous  membrane  of  the  nose.  Machnoff  also  suc- 
ceeded in  infecting  guinea-pigs  with  anthrax  through  the  unin- 
jured skin  of  the  back,  and,  as  a  result  of  subsequent  microscop 
ical  examination  of  stained  sections,  arrived  at  the  conclusion  that 
the  principal  channel  through  which  infection  was  accomplished  was 
the  hair  follicles.  Braunschweig,  in  a  series  of  experiments  in  which 
he  introduced  various  pathogenic  bacteria  into  the  conjunctival  sac 
of  mice,  rabbits,  and  guinea-pigs,  obtained  a  negative  result  with  the 
anthrax  bacillus,  the  bacillus  of  mouse  septicaemia,  the  bacillus  of 
chicken  cholera,  and  Micrococcus  tetragenus;  but  the  bacillus  ob- 
tained by  Bibbert  from  the  intestinal  diphtheria  of  rabbits  gave  a 
positive  result  in  five  mice,  two  guinea-pigs,  and  a  rabbit. 


230  CHANNELS    OF   INFECTION. 

Infection  through  the  mucous  membrane  of  the  intestine  no 
doubt  occurs  in  certain  diseases.  This  is  believed  to  be  a  common 
mode  of  the  infection  of  sheep  and  cattle  with  anthrax,  and  probably 
also  in  the  infectious  disease  of  swine  known  as  hog  cholera.  The 
anthrax  bacillus  would  be  destroyed  by  the  acid  secretions  of  the 
stomach,  but  if  spores  are  present  in  food  ingested  they  will  reach 
the  intestine.  The  experiments  of  Korkunoff  do  not,  however,  sup- 
port the  view  that  infection  is  likely  to  occur  in  this  way.  In  a  series 
of  experiments  upon  white  mice  fed  with  bread  containing  a  quantity 
of  anthrax  spores  the  result  was  uniformly  negative,  but  exception- 
ally infection  occurred  in  rabbits.  The  same  author  obtained  posi- 
tive results  in  rabbits  fed  with  food  to  which  a  pure  culture  of  the 
bacillus  of  chicken  cholera  had  been  added. 

Buchner,  in  experiments  upon  mice  and  guinea-pigs  fed  with 
material  containing  anthrax  spores,  obtained  a  positive  result  in  four 
out  of  thirty-three  animals.  This  is  no  doubt  the  usual  mode  of  in- 
fection in  typhoid  fever  in  man. 

Infection  may  also  occur  through  the  mucous  membrane  of  the 
respiratory  organs.  This  has  been  demonstrated  by  several  bac- 
teriologists, and  especially  by  the  experiments  of  Buchner,  who 
mixed  dried  anthrax  spores  with  ly  cop  odium  powder  or  pulverized 
charcoal,  and  caused  mice  and  guinea-pigs  to  respire  an  atmosphere 
containing  this  powder  in  suspension.  In  a  series  of  sixty- six  experi- 
ments fifty  animals  died  of  anthrax,  nine  of  pneumonia,  and  seven 
survived.  That  infection  did  not  occur  through  the  mucous  mem- 
brane of  the  alimentary  canal  was  proved  by  comparative  experi- 
ments in  which  animals  were  fed  with  double  the  quantity  of  spores 
used  in  the  inhalation  experiments.  Out  of  thirty- three  animals  fed 
in  this  way  but  four  contracted  anthrax.  That  infection  occurred 
through  the  lungs  was  also  demonstrated  by  the  microscopical  ex- 
amination of  sections  and  by  culture  experiments,  which  showed  that 
the  lungs  were  extensively  invaded,  while  in  many  cases  the  spleen 
contained  no  bacilli.  Positive  results  were  also  obtained  with  cul- 
tures of  the  anthrax  bacillus  not  containing  spores,  which  the  ani- 
mals were  made  to  inhale  in  the  form  of  spray.  But  in  this  case  a 
considerable  quantity  was  required,  and  a  sero-fibrinous  pneumonia 
was  usually  produced  as  well  as  general  infection;  the  inhalation  of 
small  quantities  gave  no  result.  Positive  results  in  rabbits  were  also 
obtained  by  causing  them  to  inhale  considerable  quantities  of  a  spray 
containing  the  bacillus  of  chicken  cholera. 

The  fact  that  large  quantities  of  a  liquid  culture  of  these  virulent 
bacilli  were  required  to  infect  very  susceptible  animals  by  way  of 
the  pulmonary  mucous  membrane,  and  that  Buchner  failed  to  cause 
the  infection  of  these  animals  with  small  quantities  of  a  pure  culture 


CHANNELS   OF   INFECTION.  231 

inhaled  in  the  form  of  spray,  indicates  that  this  is  not  a  common 
mode  of  infection  in  the  absence  of  spores.  This  view  receives 
further  support  from  the  experiments  of  Hildebrandt,  who  made 
tracheal  fistulse  in  three  rabbits,  and,  after  the  wound  had  entirely 
healed,  injected  into  the  trachea  of  each  a  pure  culture  of  the  anthrax 
bacillus,  which  was  proved  to  be  virulent  by  inoculation  in  mice  or 
guinea-pigs.  All  of  the  animals  remained  in  good  health.  On  the 
other  hand,  three  rabbits  which  received  in  the  same  way  a  pure  cul- 
ture of  the  bacillus  of  rabbit  septicaemia  died  as  a  result  of  general 
infection. 

That  man  may  be  infected  with  anthrax  by  way  of  the  respira- 
tory organs  seems  to  be  well  established.  In  England  the  disease 
known  as  "wool-sorter's  disease"  results  from  infection  in  this  way 
among  workmen  engaged  in  sorting  wool,  which  is  liable  to  contain 
the  spores  of  the  anthrax  bacillus  when  obtained  from  the  skin  of  an 
animal  which  has  fallen  a  victim  to  this  disease.  That  infection 
occurs  through  the  lungs  is  shown  by  the  fact  that  these  organs  are 
first  involved,  the  disease  being,  in  fact,  a  pulmonic  anthrax. 

While  these  experiments  prove  the  possibility  of  infection  through 
the  respiratory  mucous  membrane,  other  experiments  made  by  Hil- 
debrandt show  that  under  ordinary  circumstances  bacteria  suspended 
in  the  air  do  not  reach  the  trachea  in  rabbits,  but  are  deposited  upon 
the  mucous  membrane  of  the  mouth,  nares,  and  fauces.  In  healthy 
rabbits  the  tracheal  mucus  was,  as  a  rule,  found  to  be  free  from  bac- 
teria, while  they  were  very  numerous  in  mucus  obtained  from  the 
mouth  or  nares.  But  when  a  rabbit  was  made  to  inhale  for  half  an 
hour  an  atmosphere  charged  with  the  spores  of  Aspergillus  f  umigatus 
their  presence  in  the  lungs  was  demonstrated  by  cultivation,  the  ani- 
mal being  killed  for  the  purpose  half  an  hour  after  the  inhalation 
experiment. 

The  rapidity  with  which  infection  may  occur  is  shown  by  the 
experiments  of  Nissen,  Pfuhl,  and  others.  In  mice  inoculated  with 
anthrax  bacilli  at  the  tip  of  the  tail  fatal  anthrax  has  resulted, 
although  the  tail  was  amputated  ten  minutes  after  the  inoculation. 
Schimmelbusch  inoculated  fresh  wounds  with  anthrax  cultures  (in 
mice)  and  immediately  after  treated  the  wounds  with  strong  anti- 
septic solutions,  but  the  animals  succumbed  to  infection.  Cultures 
of  the  anthrax  bacillus  have  been  obtained  from  the  liver,  spleen,  and 
kidneys  half  an  hour  after  the  infection  of  an  open  wound  on  the 
surface  of  the  body  (Schimmelbnsch  and  Ricker).  The  experiments 
of  Sherrington  and  others  show  that  pathogenic  bacteria  may  escape 
by  way  of  the  kidneys  into  the  bladder,  or  through  the  liver  into  the 
gall  bladder.  But  his  experiments  indicate  that  such  escape  does  not 
occur  through  healthy  organs.  Non-pathogenic  bacteria  injected 


232  CHANNELS    OF   INFECTION. 

into  the  circulation  were  not  found  in  the  urine,  and  when  a  consid- 
erable quantity  of  a  pathogenic  species  was  injected  into  a  vein  there 
was  no  immediate  appearance  of  bacteria  in  the  urine,  but  they  were 
found  later,  probably  as  a  result  of  lesions  in  the  secreting  organ  due 
to  their  local  action  or  to  that  of  their  toxic  products.  In  man  the 
presence  of  pathogenic  bacteria  in  the  urine  has  been  frequently  veri- 
fied, especially  in  typhoid  fever,  pneumonia,  and  streptococcus  in- 
fection. When,  as  a  result  of  the  establishment  of  foci  of  infection 
in  the  liver,  localized  necrosis  of  tissue  'occurs,  the  pathogenic  bac- 
teria to  which  the  infection  is  due  escape  with  the  bile  and  enter 
the  intestine.  It  is  probable  that  escape  through  the  walls  of  the 
intestine  does  not  occur  unless  there  is  a  local  lesion  of  some  kind,  as 
in  typhoid  fever. 

The  presence  of  tubercle  bacilli  in  the  milk  of  cows  has  been 
repeatedly  demonstrated,  and  in  a  certain  proportion  of  the  cases 
they  have  been  found  in  the  milk  of  cows  whose  udders  gave 
no  evidence  of  being  the  seat  of  a  tubercular  process.  Usually,  how- 
ever, when  tubercle  bacilli  are  found  in  the  milk  the  cow's  udder 
is  already  involved  in  the  disease.  The  milk  of  women  with  puer- 
peral fever  has  been  found  to  contain  streptococci ;  and  in  mastitis 
from  a  localized  infection  by  pyogenic  cocci  these  are  found  in  the 
milk.  It  must  be  remembered,  however,  that  both  Staphylococcus 
albus  and  aureus  have  been  found  in  the  milk  of  healthy  women. 
The  micrococcus  of  pneumonia  has  been  found  in  the  milk  of  women 
suffering  from  croupous  pneumonia  (Foa,  and  Bordoni-Uffreduzzi) . 
Various  observers  (Brunner,  Tizzoni,  von  Eiselsberg)  have  reported 
the  presence  of  pus  cocci  in  the  sweat  of  patients  suffering  from  sep- 
tica3mia,  and  the  experiments  of  Brunner  indicate  that  they  may  have 
escaped  through  the  sweat  glands.  This,  however,  does  not  appear 
to  be  definitely  established. 


III. 

SUSCEPTIBILITY  AND   IMMUNITY. 

No  questions  in  general  biology  are  more  interesting,  or  more 
important  from  a  practical  point  of  view,  than  those  which  relate  to 
the  susceptibility  of  certain  animals  to  the  pathogenic  action  of  cer- 
tain species  of  bacteria,  and  the  immunity,  natural  or  acquired,  from 
such  pathogenic  action  which  is  possessed  by  other  animals.  It  has 
long  been  known  that  certain  infectious  diseases,  now  demonstrated 
to  be  of  bacterial  origin,  prevail  only  or  principally  among  animals 
of  a  single  species.  Thus  typhoid  fever,  cholera,  and  relapsing 
fever  are  diseases  of  man,  and  the  lower  animals  do  not  suffer  from 
them  when  they  are  prevailing  as  an  epidemic.  On  the  other  hand, 
man  has  a  natural  immunity  from  many  of  the  infectious  diseases  of 
the  lower  animals,  and  diseases  of  this  class  which  prevail  among 
animals  are  frequently  limited  to  a  single  species.  Again,  several 
species,  including  man,  may  be  susceptible  to  a  disease,  while  other 
animals  have  a  natural  immunity  from  it.  Thus  tuberculosis  is 
common  to  man,  to  cattle,  to  apes,  and  to  the  small  herbivorous  ani- 
mals, while  the  carnivora  are,  as  a  rule,  immune  ;  anthrax  may  be 
communicated  by  inoculation  to  man,  to  cattle,  to  sheep,  to  guinea- 
pigs,  rabbits,  and  mice,  but  the  rat,  the  dog,  carnivorous  animals,  and 
birds  are  generally  immune  ;  glanders,  which  is  essentially  a  disease 
of  the  equine  genus,  may  be  communicated  to  man,  to  the  guinea- 
pig,  and  to  field  mice,  while  house  mice,  rabbits,  cattle,  and  swine 
are  to  a  great  extent  immune. 

In  addition  to  this  general  race  immunity  or  susceptibility  we 
have  individual  differences  in  susceptibility  or  resistance  to  the  ac- 
tion of  pathogenic  bacteria,  which  may  be  either  natural  or  acquired. 
As  a  rule,  young  animals  are  more  susceptible  than  older  ones. 
Thus  in  man  the  young  are  especially  susceptible  to  scarlet  fever, 
whooping  cough,  and  other  "children's  diseases,"  and  after  forty 
years  of  age  the  susceptibility  to  tubercular  infection  is  very  much 
diminished.  Among  the  lower  animals  it  is  a  matter  of  common 
laboratory  experience  that  the  very  young  of  a  susceptible  species 
may  be  infected  when  inoculated  with  an  "attenuated  culture" 
which  older  animals  of  the  same  species  are  able  to  resist. 


234  SUSCEPTIBILITY   AMD    IMMUNITY. 

Considerable  differences  as  to  susceptibility  may  also  exist  among 
adults  of  the  same  species.  In  man  these  differences  in  individual 
susceptibility  to  infectious  diseases  are  frequently  manifested.  Of  a 
number  of  persons  exposed  to  infection  in  the  same  way,  some  may 
escape  entirely  while  others  have  attacks  differing  in  severity  and 
duration.  In  our  experiments  upon  the  lower  animals  we  constantly 
meet  with  similar  results,  some  individuals  proving  to  be  exception- 
ally resistant.  Exceptional  susceptibility  or  immunity  may  be  to 
some  extent  a  family  characteristic  or  one  of  race.  Thus  the  negro 
race  is  decidedly  less  subject  to  yellow  fever  than  the  white  race, 
and  this  disease  is  more  fatal  among  the  fair-skinned  races  of  the 
north  of  Europe  than  among  the  Latin  races  living  in  tropical  or  sub- 
tropical regions.  On  the  other  hand,  small-pox  appears  to  be  excep- 
tionally fatal  among  negroes  and  dark-skinned  races  generally. 

A  very  remarkable  instance  of  race  immunity  is  that  of  Algerian 
sheep  against  anthrax,  a  disease  which  is  very  fatal  to  other  sheep. 

In  the  instances  mentioned  race  immunity  is  probably  an  ac- 
quired tolerance  due  to  natural  selection  and  inheritance.  If,  for 
example,  a  susceptible  population  is  exposed  to  the  ravages  of  small- 
pox, the  least  susceptible  individuals  will  survive  and  may  be  the  pa- 
rents of  children  who  will  be  likely  to  inherit  the  special  bodily  char- 
acters upon  which  this  comparative  immunity  depends.  The  ten- 
dency of  continuous  or  repeated  exposure  to  the  same  pathogenic 
agent  will  evidently  be  to  establish  a  race  tolerance ;  and  there  is 
reason  to  believe  that  such  has  been  the  effect  in  the  case  of  some 
of  the  more  common  infectious  diseases  of  man,  which  have  been 
noticed  to  prevail  with  especial  severity  when  first  introduced  among 
a  virgin  population,  as  in  the  islands  of  the  Pacific,  etc. 

In  the  same  way  we  may  explain  the  immunity  which  carnivor- 
ous animals  have  for  anthrax  and  various  forms  of  septicaemia  to 
which  the  herbivora  are  very  susceptible  when  the  pathogenic  germ 
is  introduced  into  their  bodies  by  inoculation.  From  time  immemo- 
rial the  carnivora  have  been  in  the  habit  of  fighting  over  the  dead 
bodies  of  herbivorous  animals,  some  of  which  may  have  fallen  a  prey 
to  these  infectious  germ  diseases,  and  in  their  fighting  they  receive 
wounds,  inoculated  with  the  infectious  material  from  these  bodies, 
which  would  be  fatal  to  a  susceptible  animal.  If  at  any  time  in  the 
past  a  similar  susceptibility  existed  among  the  carnivora,  with  indi- 
vidual differences  as  to  resisting  power,  it  is  evident  that  there  would 
be  a  constant  tendency  for  the  most  susceptible  individuals  to  perish 
and  for  the  least  susceptible  to  survive. 

But  if  we  admit  this  to  be  a  probable  explanation  of  the  immu- 
nity of  carnivorous  animals  from  septic  infection,  we  have  not  yet 
explained  the  precise  reason  for  the  immunity  enjoyed  by  the 


SUSCEPTIBILITY   AXD    IMMUNITY.  235 

selected  individuals  and  their  progeny.  The  essential  difference  be- 
tween a  susceptible  and  immune  animal  depends  upon  the  fact  that 
in  one  the  pathogenic  germ,  when  introduced  by  accident  or  ex- 
perimental inoculation,  multiplies  and  invades  the  tissues  or  the 
blood,  where,  by  reason  of  its  nutritive  requirements  and  toxic  pro- 
ducts, it  produces  changes  in  the  tissues  arid  fluids  of  the  body  incon- 
sistent with  the  vital  requirements  of  the  infected  animal ;  while  in 
the  immune  animal  multiplication  does  not  occur  or  is  restricted  to  a 
local  invasion  of  limited  extent,  and  in  which  after  a  time  the  re- 
sources of  nature  suffice  to  destroy  the  parasitic  invader. 

Now  the  question  is,  upon  what  does  this  essential  difference  de- 
pend ?  Evidently  upon  conditions  favorable  or  unfavorable  to  the 
development  of  the  pathogenic  germ ;  or  upon  its  destruction  by 
some  active  agent  present  in  the  tissues  or  fluids  of  the  body  of  the 
immune  animal;  or  upon  a  neutralization  of  its  toxic  products  by  some 
substance  present  in  the  body  of  the  animal  which  survives  infec- 
tion. 

What,  then,  are  the  unfavorable  conditions  which  may  be  supposed 
to  prevent  development  in  immune  animals  ?  In  the  first  place,  the 
temperature  of  the  body  may  not  be  favorable.  Certain  pathogenic 
bacteria  are  only  able  to  develop  within  very  narrow  temperature  lim- 
its, and,  if  all  other  conditions  were  favorable,  could  not  be  expected 
to  multiply  in  the  bodies  of  cold-blooded  animals.  Or  the  temperature 
of  warm-blooded  animals,  and  especially  of  fowls,  may  be  above  the 
point  favorable  for  their  development.  This  is  the  explanation 
offered  by  Pasteur  of  the  immunity  of  fowls,  which  are  usually  re- 
fractory against  anthrax  ;  and  in  support  of  this  view  he  showed  by 
experiment  that  when  chickens  are  refrigerated  after  inoculation,  by 
being  partly  immersed  in  cold  water,  they  are  liable  to  become  in- 
fected and  to  perish.  But,  as  pointed  out  by  Koch,  the  sparrow, 
which  has  a  temperature  as  high  as  that  of  the  chicken,  may  con- 
tract anthrax  without  being  refrigerated.  We  must  not,  therefore, 
too  hastily  conclude  that  the  success  in  Pasteur's  experiment  de- 
pended alone  upon  a  reduction  of  the  body  heat.  Gibier  has  shown 
that  the  anthrax  bacillus  may  multiply  in  the  bodies  of  frogs  or 
fish,  if  these  are  kept  in  water  having  a  temperature  of  35°  C. 
But  the  anthrax  bacillus  grows  within  comparatively  wide  tempera- 
ture limits,  while  other  pathogenic  bacteria  are  known  to  have  a 
more  restricted  temperature  range  and  would  be  more  decidedly 
influenced  by  this  factor — e.g.,  the  tubercle  bacillus. 

The  composition  of  the  body  fluids,  and  especially  their  reaction, 
is  probably  a  determining  factor  in  some  instances.  Thus  Behring 
has  ascribed  the  failure  of  the  anthrax  bacillus  to  develop  in  the 
white  rat,  which  possesses  a  remarkable  immunity  against  anthrax, 


236  SUSCEPTIBILITY   AND    IMMUNITY. 

to  the  highly  alkaline  reaction  of  the  blood  and  tissue  juices  of  this 
animal.  Behring  claims  to  have  obtained  experimental  proof  of  the 
truth  of  this  explanation  by  feeding  white  rats  on  an  exclusively 
vegetable  diet  or  by  adding  acid  phosphate  of  lime  to  their  food,  by 
which  means  this  excessive  alkalinity  of  the  blood  is  diminished. 
Rats  so  treated  are  said  to  lose  their  natural  immunity,  and  to  die  as 
a  result  of  inoculation  with  virulent  cultures  of  the  anthrax  bacillus. 

The  experiments  of  Nuttall,  Behring,  Buchner,  and  others  have 
established  the  fact  that  recently  dratvn  blood  of  various  animals 
possesses  decided  germicidal  power,  and  Buchner  has  shown  that 
this  property  belongs  to  the  fluid  part  of  the  blood  and  not  to  its 
cellular  elements.  It  has  also  been  shown  that  aqueous  humor,  the 
fluid  of  ascites,  and  lymph  from  the  dorsal  lymph  sac  of  a  frog 
possess  the  same  power.  This  power  to  kill  bacteria  is  destroyed  by 
heat,  and  is  lost  when  the  blood  has  been  kept  for  a  considerable 
time,  but  it  is  not  neutralized  by  freezing.  Further,  this  power  to 
destroy  bacteria  differs  greatly  for  different  species,  being  very  de- 
cided in  the  case  of  certain  pathogenic  bacteria,  less  so  for  others, 
and  absent  in  the  case  of  certain  common  saprophytes.  Behring 
has  also  shown  that  the  blood  of  different  animals  differs  consider- 
ably in  this  regard,  and  that  the  blood  of  the  rat  and  of  the  frog, 
which  animals  have  a  natural  immunity  against  anthrax,  is  espe- 
cially fatal  to  the  anthrax  bacillus.  The  experiments  made  show 
that  this  germicidal  power  is  very  prompt  in  its  action,  but  that  it  is 
limited  as  to  the  number  of  bacteria  which  can  be  destroyed  by  a 
given  quantity  of  blood  serum.  When  the  number  is  excessive,  de- 
velopment occurs  after  an  interval  during  which  a  limited  destruc- 
tion has  taken  place.  It  would  appear  that  the  element  in  the  blood 
to  which  this  germicidal  action  is  due  is  neutralized  in  exercising 
this  power  ;  and  as,  independently  of  this,  blood  serum  is  an  excel- 
lent culture  medium  for  bacteria,  an  abundant  development  takes 
place  when  the  destruction  has  been  incomplete. 

Buchner  (1889)  first  proved  by  experiment  that  the  germicidal 
power  of  the  blood  of  dogs  and  rabbits  does  not  depend  upon  the 
presence  of  the  cellular  elements,  but  is  present  in  clear  serum  which 
has  been  allowed  to  separate  from  the  clot  in  a  cool  place.  Exposure 
for  an  hour  to  a  temperature  of  55°  C.  destroys  the  germicidal  action 
of  serum  as  well  as  of  blood. 

The  researches  of  Buchner,  of  Hankin,  and  others,  show  that  this 
germicidal  power  of  fresh  blood  serum  depends  upon  the  presence  of 
proteids,  to  which  the  first-named  bacteriologist  has  given  the  name 
of  "alexins."  Hankin,  in  his  paper  upon  the  origin  of  these  "defen- 
sive proteids"  in  the  animal  body  (1892),  arrives  at  the  conclusion 


SUSCEPTIBILITY   AND   IMMUNITY.  237 

that  while  they  are  present  in  the  cell-free  serum  they  are  the  prod- 
uct of  certain  leucocytes — Ehrlich's  eosinophile  cells.  He  believes 
that  the  eosinophile  granules  become  dissolved  in  the  serum  and  con- 
stitute the  germicidal  proteid  which  is  shown  to  be  present  by  ex- 
periments upon  bacteria.  According  to  Hankin  the  separation  of 
these  granules  can  be  witnessed  under  the  microscope.  They  first 
accumulate  upon  one  side  of  the  cell  and  then  gradually  disappear, 
and  as  this  occurs  a  considerable  increase  in  the  bactericidal  power 
of  the  serum  can  be  demonstrated.  The  germicidal  power  of  the 
blood  serum  is  also  said  to  be  increased  when  the  number  of  leuco- 
cytes is  considerably  augmented,  as  occurs  when  a  sterilized  culture 
of  Vibrio  Metschnikovi  is  injected  subcutaneously.  Also  by  treat- 
ment which  favors  a  separation  of  the  alexin  from  the  leucocytes, 
i.e.,  a  solution  of  the  eosinophile  granules.  This  may  be  accom- 
plished by  the  injection  of  an  extract  of  the  thymus  gland  of  the 
calf,  or  by  simply  allowing  the  drawn  blood  to  stand  for  several  hours 
at  a  temperature  of  38°  to  40°  C. 

Buchner's  latest  communication  upon  the  subject  shows  that  he 
also  attributes  the  origin  of  the  germicidal  proteid  in  fresh  blood 
serum  to  the  leucocytes.  In  his  paper  on  "Immunity,"  read  at  the 
Eighth  International  Congress  on  Hygiene  and  Demography  (Buda- 
pest, 1894),  he  calls  attention  in  the  first  place  to  the  fact  that  a 
clearly  marked  distinction  must  be  made  between  natural  immunity 
and  acquired  immunity,  inasmuch  as  the  "  alexins  "  and  "  antitoxins  " 
have  very  different  properties.  The  first-mentioned  proteid s  are  de- 
stroyed by  a  comparatively  low  temperature  (55°  to  60°  C.),  while  the 
antitoxins  resist  a  considerably  higher  temperature,  and,  unlike  the 
alexins,  have  no  bactericidal  or  globulicidal  action.  A  very  remark- 
able fact  developed  in  Buchner's  experiments  is  that  the  blood  serum 
from  the  dog  and  from  the  rabbit,  when  mixed,  neutralize  each  other 
so  far  as  their  germicidal  power  is  concerned. 

By  injecting  sterilized  emulsions  of  wheat-flour  paste  in  the 
pleural  cavity  of  rabbits  and  dogs  Buchner  succeeded  in  obtaining  an 
exudate  which  had  more  decided  germicidal  power  than  the  blood 
or  serum  of  the  same  animal.  This  was  evidently  due  to  the  large 
number  of  leucocytes  present,  but  not  to  their  phagocytic  action,  as 
was  shown  by  experiment.  By  freezing  the  exudate  the  leucocytes 
were  killed,  but  the  germicidal  action  of  the  fluid  was  rather  in- 
creased than  diminished  by  freezing.  While  freezing  had  no  effect 
upon  the  germicidal  action  of  the  pleural  exudate,  this  was  always 
neutralized  by  exposure  to  a  temperature  of  55°  C. 

Emmerich,  Tsuboi,  Steinmetz,  and  Low  (1892),  as  a  result  of  ex- 
tended experiments,  arrived  at  the  conclusion  that  the  germicidal 
action  of  blood  serum  "  depends  upon  a  specific  property  of  the  alkali 


238  SUSCEPTIBILITY   AND    IMMUNITY. 

serumalbumin,  and  that  it  is  a  purely  chemical  process."  They 
state  that  when  the  germicidal  power  is  neutralized  by  heat  it  may 
be  restored  by  the  addition  of  an  alkali.  Buclmer  repeated  the  ex- 
periments of  Emmerich  and  his  associates  and  obtained  similar  re- 
sults, but  interprets  them  differently.  According  to  him  the  serum 
does  not  regain  its  germicidal  power,  but  after  the  addition  of  an 
alkali  and  subsequent  dialyzing  the  nutritive  value  of  the  serum  is  so 
diminished  that  the  bacteria  do  not  develop  in  it. 

Pane  (1892)  has  made  experiments  which  give  additional  weight 
to  the  assumption  that  the  alkalinity  of  the  blood  is  an  important 
factor  in  accounting  for  immunity.  He  states  that  carbonate  of 
soda,  dissolved  in  water,  in  the  proportion  of  1:3,000,  has  a  de- 
cided germicidal  action  upon  the  anthrax  bacillus,  equal  to  that  of 
the  blood  serum  of  the  rabbit.  And  that  when  rabbit  serum  is  com- 
pletely neutralized  it  no  longer  has  any  injurious  action  on  anthrax 
bacilli. 

Zagari  and  Innocente  (1892)  also  arrived  at  the  conclusion  that 
the  diminished  resistance  to  anthrax  infection  resulting  from  curare 
poisoning  in  frogs,  and  from  chloral  or  alcohol  in  dogs  (Platania),  in 
fowls  as  a  result  of  starvation  (Canalis  and  Morpurgo),  in  white 
mice  as  a  result  of  fatigue  (Charin  and  Roger) ,  is,  in  fact,  due  to 
diminished  alkalinity  of  the  blood,  which  they  found  to  correspond 
with  the  increased  susceptibility  resulting  from  the  causes  men- 
tioned. 

Buchner  (1892)  states  that  several  of  the  ammonium  salts,  and 
especially  ammonium  sulphate,  cause  an  increase  in  the  germicidal 
action  of  blood  serum,  and  also  increase  its  resistance  to  the  neutral- 
izing effects  of  heat.  The  experiments  of  Pansini  and  Calabrese 
(1894)  show,  on  the  contrary,  that  the  addition  of  uric  acid  to  blood 
serum  diminishes  its  bactericidal  activity,  as  does  also  the  presence 
of  glucose.  That  certain  infectious  diseases  are  especially  virulent 
in  persons  suffering  from  diabetes  is  a  frequently  repeated  clinical 
observation. 

Van  Fodor  has  shown  by  experiment  that  the  injection  of  an 
alkali  into  the  circulation  of  a  rabbit  increases  its  resistance  to 
anthrax  infection  and  the  germicidal  activity  of  its  blood  serum. 
The  same  bacteriologist  has  found  that  when  a  rabbit  is  infected 
with  anthrax,  the  alkalinity  of  its  blood  is  notably  increased  during 
the  first  twenty-four  hours,  when  we  may  suppose  that  the  powers 
of  nature  are  brought  to  bear  to  resist  the  invading  parasite,  and  that 
after  this  time  it  rapidly  diminishes.  Ten  hours  after  infection  (by 
subcutaneous  inoculation?)  the  alkalinity  of  the  blood  had  increased 
21.5  per  cent.  Shortly  before  the  death  of  the  animal  a  diminution 
of  26.3  per  cent  was  noted.  This  diminution  was  observed  in  thirty- 


SUSCEPTIBILITY    AND    IMMUNITY.  239 

four"  out  of  thirty-nine  animals  experimented  upon,  and  these  ani- 
mals succumbed  to  the  anthrax  infection  in  a  shorter  time  than  did 
the  other  five  in  which  there  was  no  such  diminution. 

It  seems  probable  that  the  germicidal  property  of  freshly  drawn 
blood  serum  is  not  due  to  its  alkalinity,  per  se,  but  to  the  fact  that 
the  germicidal  constituent  is  only  soluble  in  an  alkaline  fluid.  The 
researches  of  Vaughn,  McClintock,  and  Novy  indicate  that  this  ger- 
micidal constituent  is  a  nuclein.  Dr.  Vaughn,  in  his  last  published 
paper  upon  "Nucleins  and  Nuclein  Therapy,"  says:  "Kossel,  of 
Berlin,  has  confirmed  our  statements  concerning  the  germicidal 
action  of  the  nucleins.  Dr.  McClintock  and  I  have  also  demon- 
strated that  the  germicidal  constituent  of  blood  serum  is  a  nuclein. 
This  nuclein  is  undoubtedly  furnished  by  the  polynuclear  white 
corpuscles."  Denys  has  (1894)  reported  the  results  of  experi- 
ments made  in  his  laboratory  by  Van  der  Velde,  which  give  sup- 
port to  the  conclusion  reached  by  Vaughn.  In  these  experiments  a 
sterilized  culture  of  staphylococci  was  injected  into  the  pleural  cavity 
of  rabbits  in  order  to  obtain  an  exudate.  At  intervals  of  two  hours 
this  exudate  was  obtained  by  killing  one  of  the  animals  in  the  series 
experimented  upon,  and  at  the  same  time  blood  from  the  animal  was 
secured.  Both  the  exudate  and  the  blood  were  placed  in  a  centrifugal 
machine,  in  order  to  obtain  a  serum  free  from  corpuscular  elements. 
The  germicidal  activity  of  the  serum  was  then  tested.  The  general 
result  of  the  experiments  was  to  show  that  the  longer  the  interval 
after  the  injection  into  the  pleural  cavity  the  more  potent  the  ger- 
micidal activity  of  the  exudate  became,  and  that  there  was  no  corre- 
sponding increase  in  the  activity  of  the  blood  serum  obtained  from 
the  circulation.  At  the  end  of  ten  or  twelve  hours,  the  serum  from 
the  exndate  killed  all  of  the  staphylococci  in  a  bouillon  culture  twenty 
times  as  great  in  quantity  as  the  germicidal  serum  used  in  the  ex- 
periment. The  absence  of  any  increase  in  germicidal  power  in  the 
blood  serum  taken  from  the  general  circulation  shows  that  the  nota- 
ble increase  manifested  by  the  exudate  was  due  to  local  causes ;  and 
as  a  matter  of  fact  it  corresponded  with  an  increase  in  the  number  of 
leucocytes  as  found  in  the  pleural  exudate. 

Thus  it  will  be  seen  that  the  independent  researches  of  Hankin, 
of  Buchner,  of  Vaughn,  and  of  other  competent  bacteriologists,  have 
led  them  to  the  same  ultimate  result  so  far  as  the  origin  of  the  ger- 
micidal constituent  of  the  blood  is  concerned,  and  that  the  leucocytes 
appear  to  play  an  important  role  in  the  protection  of  the  animal  body 
from  invasion  by  bacteria  (natural  immunity). 

It  has  been  shown  by  several  investigators  that  the  number  of 
leucocytes  increases  in  certain  infectious  diseases,  and  this  increase, 
together  with  an  increased  alkalinity  of  the  blood,  which  has  here- 


240  SUSCEPTIBILITY    AND    IMMUNITY. 

tofore  been  referred  to,  appears  to  be  a  provision  of  nature  for  over- 
coming the  infection  which  has  already  occurred. 

It  has  been  demonstrated  by  experiment  that  naturally  immune 
animals  may  be  infected  by  the  addition  of  certain  substances  to  cul- 
tures of  pathogenic  bacteria.  Thus  Arloing  was  able  to  induce  symp- 
tomatic anthrax  in  animals  naturally  immune  for  this  disease  by 
mixing  with  his  cultures  various  chemical  substances,  such  as  car- 
bolic acid,  pyrogallic  acid,  and  especially  lactic  acid  (twenty  per 
cent).  Leo  has  shown  that  white  mice,  which  are  not  subject  to 
the  pathogenic  action  of  the  glanders  bacillus,  may  be  rendered  sus- 
ceptible by  feeding  them  for  some  time  upon  phloridzin,  which  gives 
rise  to  an  artificial  diabetes,  and  causes  the  tissues  to  become  im- 
pregnated with  sugar. 

Bouchard  has  found  that  very  small  doses  of  a  pure  culture  of 
Bacillus  pyocyaneus  are  fatal  to  rabbits  when  at  the  same  time  a 
considerable  quantity  of  a  filtered  culture  of  the  same  bacillus  is  in- 
jected into  a  vein.  The  animal  could  have  withstood  the  filtered 
culture  alone,  or  the  bacillus  injected  beneath  its  skin ;  but  its  resist- 
ing power — natural  immunity — is  overcome  by  the  combined  action 
of  the  living  bacilli  and  the  toxic  substances  contained  in  the  filtered 
culture.  The  same  result  may  be  obtained  by  injecting  sterilized 
cultures  of  a  different  microorganism.  Thus  Roger  has  shown  that 
the  rabbit,  which  has  a  natural  immunity  against  symptomatic 
anthrax,  succumbs  to  infection  when  inoculated  with  a  culture  of  the 
bacillus  of  this  disease,  if  at  the  same  time  it  receives  an  injection  of 
a  sterilized  or  non-sterilized  culture  of  Bacillus  prodigiosus.  Monti 
has  succeeded  in  killing  animals  with  old  and  attenuated  cultures  of 
Streptococcus  pyogenes,  or  of  Staphylococcus  pyogenes  aureus,  by  in- 
jecting at  the  same  time  a  culture  of  Proteus  vulgaris.  In  a  similar 
way,  it  seems  probable,  the  normal  resistance  of  man  to  infection  by 
certain  pathogenic  bacteria  may  be  overcome.  Thus  when  water 
contaminated  by  the  presence  of  the  typhoid  bacillus  is  used  for 
drinking  by  the  residents  of  a  certain  town  or  district,  not  all  of 
those  who  in  this  way  are  exposed  to  infection  contract  typhoid 
fever;  and  among  those  who  do,  there  is  good  reason  to  believe  that, 
in  certain  cases  at  least,  the  result  depends  upon  an  additional  factor 
of  the  kind  suggested  by  the  above-mentioned  experiments — e.g.,  the 
consumption  of  food  containing  putrefactive  products,  or  the  respi- 
ration of  an  atmosphere  containing  volatile  products  of  putrefaction. 

The  natural  immunity  of  healthy  animals  may  also  be  neutralized 
by  other  agencies  which  have  a  depressing  effect  upon  the  vital  re- 
sisting power.  Thus  Nocard  and  Roux  found  by  experiment  that  an 
attenuated  culture  of  the  anthrax  bacillus,  which  was  not  fatal  to 
guinea-pigs,  killed  these  animals  when  injected  into  the  muscles  of 


SUSCEPTIBILITY   AND    IMMUNITY.  241 

the  thigh  after  they  had  been  bruised  by  mechanical  violence. 
Abarrin  and  Roger  found  that  white  rats,  which  are  not  susceptible 
to  anthrax,  became  infected  and  frequently  died  if  they  were  ex- 
hausted, previous  to  inoculation,  by  being  compelled  to  turn  a  revolv- 
ing wheel  for  a  considerable  time.  Pasteur  found  that  fowls,  which 
have  a  natural  immunity  against  anthrax,  become  infected  and 
perish  if  they  are  subjected  to  artificial  refrigeration  after  inocula- 
tion. This  has  been,  confirmed  by  the  more  recent  experiments  of 
Wagner  (1891).  According  to  Canalis  and  Morpurgo,  pigeons 
which  are  enfeebled  by  inanition  eaily  contract  anthrax  as  a  result 
of  inoculation.  Arloing  states  that  sheep  which  have  been  freely 
bled  contract  anthrax  more  easily  than  others;  and  Serafini  found 
that  when  dogs  were  freely  bled  the  bacillus  of  Friedlander,  injected 
into  the  trachea  or  the  pleural  cavity,  entered,  and  apparently  mul- 
tiplied to  some  extent  in  the  blood,  whereas  without  such  previous 
bleeding  they  were  not  to  be  found  in  the  circulating  fluid.  Certain 
anesthetic  agents  have  been  shown  also  to  produce  a  similar  result. 
Platariia  communicated  anthrax  to  immune  animals — dogs,  frogs, 
pigeons — by  bringing  them  under  the  influence  of  curare,  chloral,  or 
alcohol;  and  Wagner  obtained  similar  results  in  his  experiments 
upon  pigeons  to  which  he  had  administered  chloral.  In  man,  clini- 
cal experience  shows  that  those  who  are  addicted  to  the  excessive  use 
of  alcohol  are  especially  liable  to  contract  certain  infectious  diseases 
— pneumonia,  eiysipelas,  yellow  fever,  etc. 

The  micrococcus  of  pneumonia  is  habitually  present  in  the  sali- 
vary secretions  of  many  healthy  individuals,  and  it  is  evident  that 
an  attack  of  pneumonia  does  not  depend  alone  upon  the  presence  of 
this  micrococcus,  which  has,  nevertheless,  been  conclusively  shown 
to  be  the  usual  infectious  agent  in  cases  of  croupous  pneumonia.  No 
doubt  the  introduction  of  the  pathogenic  micrococcus  to  the  vulner- 
able point — the  lungs — is  an  essential  factor  in  the  development  of  a 
case  of  pneumonia,  but  there  is  reason  to  believe  that  there  are  other 
factors  equally  essential.  Thus  it  is  well  known  that  an  attack  of 
pneumonia  often  results  from  exposure  to  cold,  which  may  act  as  an 
exciting  cause;  and,  also,  that  a  recent  attack  of  an  acute  febrile 
disease — especially  measles — constitutes  a  predisposing  cause.  It  is 
generally  recognized  that  malnutrition,  want  of  exercise,,  insanitary 
surroundings,  and  continued  respiration  of  an  atmosphere  loaded 
with  dust,  as  in  cotton  mills,  or  a  recent  attack  of  pneumonia,  con- 
stitute predisposing  causes  to  tubercular  infection  by  way  of  the 
lungs. 

While  natural  immunity  may  be  overcome  by  the  various  depress- 
ing agencies  referred  to,  it  is  also  true  that  it  has  only  a  relative 
value  in  the  absence  of  these  predisposing  causes,  and  may  be  over- 
16 


242  SUSCEPTIBILITY    AND    IMMUNITY. 

come  by  unusual  virulence  of  the  pathogenic  infectious  agent,  or  by 
the  introduction  into  the  body  of  an  excessive  amount  of  a  pure  cul- 
ture of  the  same. 

The  pathogenic  potency  of  known  disease  germs  varies  as  widely 
as  does  the  susceptibility  of  individuals  to  their  specific  action.  In 
general  it  may  be  said  that  the  more  recently  the  germ  comes  from 
a  developed  case  of  the  disease  to  which  it  gives  rise  the  more  viru- 
lent it  is,  and  the  longer  it  has  been  cultivated  outside  of  the  animal 
body  the  more  attenuated  is  its  pathogenic  power.  Thus  when  the 
discharges  of  a  typhoid  fever  patient  find  their  way  directly  to  a 
water-supply  of  limited  amount  a  large  proportion  of  those  who 
drink  the  water  are  likely  to  be  attacked ;  but  when  a  considerable 
interval  of  time  has  elapsed  since  the  contamination  occurred, 
although  the  germs  may  still  be  present,  the  liability  to  attack  is 
much  less  on  account  of  diminished  pathogenic  virulence. 

The  development  of  an  attack  also  depends,  to  some  extent,  upon 
the  number  of  germs  introduced  into  a  susceptible  individual  at  one 
time.  The  resources  of  nature  may  be  sufficient  to  dispose  of  a  few 
bacilli,  while  a  large  number  may  overwhelm  the  resisting  power  of 
the  individual. 

The  experiments  of  Cheyne  (1886)  show  that  in  the  case  of  very 
pathogenic  species  a  single  bacillus,  or  at  least  a  very  small  number, 
introduced  beneath  the  skin,  may  produce  fatal  infection  in  a  very 
susceptible  animal,  while  greater  numbers  are  required  in  those  less 
susceptible.  Thus  a  guinea-pig  succumbed  to  general  infection  after 
being  inoculated  subcutaneously  with  anthrax  blood  diluted  to  such 
an  extent  that,  by  estimation,  only  one  bacillus  was  present  in  the 
fluid  injected ;  and  a  similar  result  was  obtained  in  mice  with  Bacillus 
murisepticus.  In  the  case  of  the  microbe  of  fowl  cholera  (Bacillus 
septicaemia  hemorrhagica}) ,  Cheyne  found  that  for  rabbits  the  fatal 
dose  was  300,000  or  more,  that  from  100,000  to  30,000  cause  a  local 
abscess,  and  that  less  than  10,000  produce  no  appreciable  effect.  The 
common  saprophyte,  Proteus  vulgaris,  was  found  to  be  pathogenic 
for  rabbits  when  injected  into  the  dorsal  muscles  in  sufficient  num- 
bers. But,  according  to  the  estimates  made,  225,000,000  were  re- 
quired to  cause  death,  while  doses  of  from  9,000,000  to  112,000,000 
produced  a  local  abscess,  and  less  than  9,000,000  gave  an  entirely 
negative  result. 

ACQUIRED   IMMUNITY. 

It  has  long  been  known  that,  in  a  considerable  number  of  infec- 
tious diseases,  a  single  attack,  however  mild,  affords  protection 
against  subsequent  attacks  of  the  same  disease;  that  in  some  cases 
this  protection  appears  to  be  permanent,  lasting  during  the  life  of  the 


SUSCEPTIBILITY   AND   IMMUNITY.  243 

individual;  that  in  others  it  is  more  or  less  temporary,  as  shown  by 
the  occurrence  of  a  subsequent  attack. 

The  protection  afforded  by  a  single  attack  not  only  differs  in  dif- 
ferent diseases,  but  in  the  same  disease  varies  greatly  in  different 
individuals.  Thus  certain  individuals  have  been  known  to  suffer 
several  attacks  of  small-pox  or  of  scarlet  fever,  although,  as  a  rule,  a 
single  attack  is  protective.  Exceptional  susceptibility  or  insuscepti- 
bility may  be  not  only  an  individual  but  a  family  characteristic,  or 
it  may  belong  to  a  particular  race. 

In  those  diseases  in  which  second  attacks  are  not  infrequent,  as, 
for  example,  in  pneumonia,  in  influenza,  or  in  Asiatic  cholera,  it  is 
difficult  to  judge  from  clinical  experience  whether  a  first  attack  exerts 
any  protective  influence.  But  from  experiments  upon  the  lower  ani- 
mals we  are  led  to  believe  that  a  certain  degree  of  immunity,  lasting 
for  a  longer  or  shorter  time,  is  afforded  by  an  attack  of  pneumonia 
or  of  cholera,  and  probably  of  all  infectious  diseases  due  to  bacterial 
parasites.  In  the  malarial  fevers,  which  are  due  to  a  parasite  of  a 
different  class,  one  attack  affords  no  protection,  but  rather  predis- 
poses to  a  subsequent  attack. 

In  those  diseases  in  which  a  single  attack  is  generally  recognized 
as  being  protective,  exceptional  cases  occur  in  which  subsequent 
attacks  are  developed  as  a  result  of  unusual  susceptibility  or  expo- 
sure under  circumstances  especially  favorable  to  infection.  Maiselis 
(1894)  has  gone  through  the  literature  accessible  to  him  for  the 
purpose  of  determining  the  frequency  with  which  second  attacks 
occur  in  the  various  diseases  below  mentioned.  The  result  is  as 
follows : 

Second  Third         Fourth  „,  .   , 

Attacks.         Attacks.      Attacks.  M- 

Small-pox 505  9  0  514 

Scarlet  fever 29  4  0  33 

Measles 36  1  0  37 

Typhoid  fever 202  5  1  208 

Cholera 29  3  2  34 

These  figures  support  the  view  generally  entertained  by  physi- 
cians that  second  attacks  of  scarlet  fever  and  of  measles  are  compar- 
atively rare,  while  second  attacks  of  small-pox  are  not  infrequently 
observed.  Considering  the  very  large  number  of  cases  of  typhoid 
fever  which  occur  annually  in  all  parts  of  Europe  and  America,  the 
number  of  second  attacks  collected  does  not  bear  a  very  large  propor- 
tion to  the  total  number  taken  sick,  although  the  recorded  cases,  of 
course,  fall  far  short  of  the  total  number  of  second  attacks  of  this 
and  the  other  diseases  mentioned. 

The  second  attacks  of  cholera  recorded  are  not  numerous,  and,  no 
doubt,  a  carefullly  conducted  investigation  made  in  the  areas  of  en- 


244  SUSCEPTIBILITY   AND    IMMUNITY. 

demic  prevalence  of  this  disease  would  show  that  second  attacks  are 
more  common  than  is  indicated  by  these  figures. 

That  immunity  may  result  from  a  comparatively  mild  attack  as 
well  as  from  a  severe  one  is  a  matter  of  common  observation  in  the 
case  of  small-pox,  scarlet  fever,  yellow  fever,  etc. ;  and  since  the  dis- 
covery of  Jenner  we  have  in  vaccination  a  simple  method  of  produc- 
ing immunity  in  the  first-mentioned  disease.  The  acquired  immunity 
resulting  from  vaccination  is  not,  however,  as  complete  or  as  per- 
manent as  that  which  results  from  an  attack  of  the  disease. 

These  general  facts  relating  to  acquired  immunity  from  infectious 
diseases  constituted  the  principal  portion  of  our  knowledge  with  re- 
ference to  this  important  matter  up  to  the  time  that  Pasteur  (1880) 
demonstrated  that  in  the  disease  of  fowls  known  as  chicken  cholera, 
which  he  had  proved  to  be  due  to  a  specific  microorganism,  a  mild 
attack  followed  by  immunity  may  be  induced  by  inoculation  with  an 
"  attenuated  virus"  —  i.e.,  by  inoculation  with  a  culture  of  the  patho- 
genic microorganism  the  virulence  of  which  had  been  so  modified 
that  it  gave  rise  to  a  comparatively  mild  attack  of  the  disease  in 
question.  Pasteur's  original  method  of  obtaining  an  attenuated  virus 
consisted  in  exposing  his  cultures  for  a  considerable  time  to  the  ac- 
tion of  atmospheric  oxygen.  It  has  since  been  ascertained  that  the 
same  result  is  obtained  with  greater  certainty  by  exposing  cultures 
for  a  given  time  to  a  temperature  slightly  below  that  which  would 
destroy  the  vitality  of  the  pathogenic  microorganism,  and  also  by  ex- 
posure to  the  action  of  certain  chemical  agents. 

Pasteur  at  once  comprehended  the  importance  of  his  discovery, 
and  inferred  that  what  was  true,  of  one  infectious  germ  disease  was 
likely  to  be  true  of  others.  Subsequent  researches,  by  this  savant 
and  by  other  bacteriologists,  have  justified  this  anticipation,  and  the 
demonstration  has  already  been  made  for  a  considerable  number  of 
similar  diseases — anthrax,  symptomatic  anthrax,  rouget. 

A  virus  which  has  been  attenuated  artificially — by  heat,  for  ex- 
ample— may  be  cultivated  through  successive  generations  without  re- 
gaining its  original  virulence.  As  this  virulence  depends,  to  a  con- 
siderable extent  at  least,  upon  the  formation  of  toxic  products  during 
the  development  of  the  pathogenic  microorganism,  we  naturally  infer 
that  diminished  virulence  is  due  to  a  diminished  production  of  these 
toxic  substances. 

There  is  reason  to  believe  that  a  natural  attenuation  of  virulence 
may  occur  in  pathogenic  bacteria  which  are  able  to  lead  a  sapro- 
phytic  existence  during  their  multiplication  external  to  the  bodies  of 
living  animals,  and  the  comparatively  mild  character  of  some  epi- 
demics is  probably  due  to  this  fact. 


SUSCEPTIBILITY   AND   IMMUNITY.  245 

Again,  cultivation  within  the  body  of  a  living  animal  may,  in 
certain  cases,  cause  a  diminution  in  the  virulence  of  a  pathogenic 
microorganism.  Thus  Pasteur  and  Thuiller  have  shown  that  the 
microbe  of  rouget  when  inoculated  into  a  rabbit  kills  the  animal,  but 
that  its  pathogenic  virulence  is  nevertheless  so  modified  that  a  cul- 
ture made  from  the  blood  of  a  rabbit  killed  by  it  is  a  suitable  "  vac- 
cine "  for  the  pig. 

On  the  other  hand,  we  have  experimental  evidence  that  the  viru- 
lence of  attenuated  cultures  may  be  reestablished  by  passing  them 
through  the  bodies  of  susceptible  animals.  Thus  a  culture  of  the 
bacillus  of  rouget,  attenuated  by  having  been  passed  through  the 
body  of  a  rabbit,  is  restored  to  its  original  virulence  by  passing  it 
through  the  bodies  of  pigeons.  And  a  culture  of  the  anthrax  bacillus 
which  will  not  kill  an  adult  guinea-pig  may  be  fatal  to  a  very  young 
animal  of  the  same  species  or  to  a  mouse,  and  the  bacillus  cultivated 
from  the  blood  of  such  an  animal  will  be  found  to  have  greatly  in- 
creased virulence. 

In  Pasteur's  inoculations  against  anthrax  "attenuated"  cultures 
are  employed  which  contain  the  living  pathogenic  germ  as  well  as 
the  toxic  products  developed  during  its  growth.  Usually  two  inocu- 
lations are  made  with  cultures  of  different  degrees  of  attenuation— 
that  is  to  say,  with  cultures  in  which  the  toxic  products  are  formed 
in  less  amount  than  in  virus  of  full  power.  The  most  attenuated 
virus  is  first  injected,  and  after  some  time  the  second  vaccine,  which 
if  injected  first  might  have  caused  a  considerable  mortality.  The 
animal  is  thus  protected  from  the  pathogenic  action  of  the  most 
virulent  cultures. 

Now,  it  has  been  shown  by  recent  experiments  that  a  similar  im- 
munity may  result  from  the  injection  into  a  susceptible  animal  of  the 
toxic  products  contained  in  a  virulent  culture,  independently  of  the 
living  bacteria  to  which  they  owe  their  origin.  Chauveau,  in  1880, 
ascertained  that  if  pregnant  ewes  are  protected  against  anthrax  by 
inoculation  with  an  attenuated  virus,  their  lambs,  when  born,  also 
give  evidence  of  having  acquired  an  immunity  from  the  disease.  As 
the  investigations  of  Davaine  seemed  to  show  that  the  anthrax 
bacillus  cannot  pass  through  the  placenta  from  the  mother  to  the 
foetus,  the  inference  seemed  justified  that  the  acquired  immunity  of 
the  latter  was  due  to  some  soluble  substance  which  could  pass  the 
placental  barrier.  More  recent  researches  by  Strauss  and  Chamber- 
lain, Malvoz  and  Jacquet,  and  others,  show  that  the  placenta  is  not 
such  an  impassable  barrier  for  bacteria  as  was  generally  believed  at 
the  time  of  Chauveau's  experiments,  so  that  these  cannot  be  accepted 
as  establishing  the  inference  referred  to.  But,  as  stated,  we  have 
more  recent  experimental  evidence  which  shows  that  immunity  may 


246  SUSCEPTIBILITY   AND   IMMUNITY. 

result  from  the  introduction  into  the  bodies  of  susceptible  animals  of 
the  toxic  substances  produced  by  certain  pathogenic  bacteria.  The 
first  satisfactory  experimental  evidence  of  this  important  fact  was 
obtained  by  Salmon  and  Smith  in  1880,  who  succeeded  in  making 
pigeons  immune  from  the  pathogenic  effects  of  cultures  of  the  bacil- 
lus of  hog  cholera  by  inoculating  them  with  sterilized  cultures  of 
this  bacillus.  In  1888  Roux  reported  similar  results  obtained  by  in- 
jecting into  susceptible  animals  sterilized  cultures  of  the  anthrax 
bacillus.  Behring  and  Kitasato,  in  1890,  reported  their  success  in 
establishing  immunity  against  virulent  cultures  of  the  bacillus  of 
tetanus  and  the  diphtheria  bacillus  by  inoculating  susceptible  ani- 
mals with  filtered,  germ-free  cultures  of  these  pathogenic  bacteria. 

In  1892  Behring,  Kitasato,  and  Wassermann  published  the  re- 
sults of  interesting  experiments  with  a  bouillon  made  from  the 
thymus  gland  of  the  calf.  They  found  that  the  tetanus  bacillus  cul- 
tivated in  this  bouillon  did  not  form  spores  and  had  comparatively 
little  virulence.  Mice  or  rabbits  inoculated  with  it  in  small  doses — • 
0.001  to  0.2  cubic  centimetre  for  a  mouse — proved  to  be  subsequently 
immune.  And  the  blood  serum  of  an  immune  rabbit  injected  into 
the  peritoneal  cavity  of  a  mouse — 0.1  to  0.5  cubic  centimetre — was 
found  to  give  it  immunity  from  the  pathogenic  action  of  a  virulent 
culture  of  the  tetanus  bacillus.  Similar  results  were  obtained  with 
several  other  pathogenic  bacteria  cultivated  in  the  thymus  bouillon — 
spirillum  of  cholera,  bacillus  of  diphtheria,  typhoid  bacillus.  We 
give  here  the  directions  for  preparing  the  thymus  bouillon  as  used  by 
the  authors  named : 


Two  or  three  thymus  glands  are  chopped  into  small  pieces  immediately 
after  they  are  taken  from  the  animal.  An  equal  part  ot  distilled  wat^r  is 
added  to  the  mass  and  stirred  for  some  time  ;  it  is  then  placed  in  an  ice  chest 
for  twelve  hours.  The  juices  are  now  expressed  through  gauze  by  means  of 
a  flesh  press.  A  clouded,  slimy  fluid  is  obtained,  which  constitutes  a  stock 
solution.  This  is  diluted  with  water,  and  a  certain  quantity  of  carbonate  of 
soda  is  added  to  the  solution  before  sterilization.  By  this  means  coagulation 
and  precipitation  of  the  active  substance  from  the  thymus  gland  are  avoided. 
The  exact  amount  of  water  and  of  sodium  carbonate  required  to  prevent  pre- 
cipitation must  be  determined  by  experiment,  as  it  differs  for  different  glands. 
Usually  an  equal  portion  of  water  and  sufficient  soda  solution  to  turn  litmus 
paper  feebly  blue  will  give  the  desired  result.  The  liquid  is  now  heated  in 
a  large  flask,  which  is  left  for  fifteen  minutes  in  the  steam  sterilizer.  The 
liquid  is  allowed  to  cool  and  then  filtered  through  fine  linen  to  remove  any 
suspended  coagula  ;  the  filtrate  has  a  milky  opalescence.  It  is  now  placed 
in  test  tubes  and  again  sterilized.  The  active  principle  is  precipitated  by  the 
addition  of  a  few  drops  of  acetic  acid . 


In  Pasteur's  inoculations  against  hydrophobia,  made  subsequently 
to  infection  by  the  bite  of  a  rabid  animal,  an  attenuated  virus  is  in- 


SUSCEPTIBILITY    AND    IMMUNITY.  247 

troduced  upon  the  surface  of  the  brain,  and  immunity  is  established 
during  the  interval — so-called  period  of  incubation — which  usually 
occurs  between  the  date  of  infection  and  the  development  of  the 
disease.  That  the  immunity  in  this  case  also  depends  upon  the 
introduction  of  a  chemical  substance  present  in  the  desiccated  spinal 
cord  of  rabbits  which  have  succumbed  to  rabies,  which  is  used  in 
these  inoculations,  is  extremely  probable.  But,  as  the  germ  of  rabies 
has  not  been  isolated  or  cultivated  artificially,  this  has  not  yet  been 
demonstrated.  Wooldridge  claims  to  have  made  susceptible  animals 
immune  against  anthrax  by  inoculating  them  with  an  aqueous  ex- 
tract of  the  testicle  or  of  the  thymus  gland  of  healthy  animals. 

We  may  mention  also  the  interesting  results  obtained  by  Em- 
merich, Freudenreich,  and  others,  who  have  shown  that  an  anthrax 
infection  in  a  susceptible  animal  inoculated  with  a  virulent  culture 
may  be  made  to  take  a  modified  and  non-fatal  course  by  the  simul- 
taneous or  subsequent  inoculation  of  certain  other  non-pathogenic 
bacteria — streptococcus  of  erysipelas,  Bacillus  pyocyaneus. 

In  a  series  of  experiments  made  by  the  writer  some  years  ago 
evidence  was  obtained  that,  under  certain  circumstances,  immunity 
from  the  effects  of  one  pathogenic  bacillus  may  be  obtained  by  the 
previous  injection  of  a  pure  culture  of  a  different  species.  In  the 
experiments  referred  to  injections  into  the  cavity  of  the  abdomen  of 
a  culture  of  Bacillus  pyocyaneus  protected  rabbits  from  the  lethal 
effects  of  Bacillus  cuniculicida  Havaniensis,  when  subsequently  in- 
jected into  the  cavity  of  the  abdomen  in  such  amount  (one  cubic 
centimetre  of  a  bouillon  culture)  as  invariably  proved  fatal  in  rabbits 
not  protected  by  such  injections. 

Before  considering  the  theories  which  have  been  offered  in  expla- 
nation of  acquired  immunity  it  is  desirable  to  call  attention  to  certain 
observations  which  have  been  made  during  the  past  few  years  relat- 
ing to  "  chemiotaxis. " 

The  term  chemiotaxis  was  first  used  by  Pfeffer  to  designate  the 
property,  observed  by  himself  and  others,  which  certain  living  cells 
exhibit  with  reference  to  non-living  organic  material,  and  by  virtue 
of  which  they  approach  or  recede  from  certain  substances.  The 
chemiotaxis  is  said  to  be  positive  when  the  living  cell  approaches,  and 
negative  when  it  recedes  from,  a  chemical  substance.  As  examples 
of  this  we  may  mention  the  approach  of  motile  bacteria  to  nutrient 
material  or  to  the  surface  of  a  liquid  medium  where  they  find  the 
oxygen  required  for  their  vital  activities  ;  and  of  leucocytes  to  cer- 
tain substances  when  these  are  introduced  beneath  the  skin  of  warm- 
er cold-blooded  animals.  This  subject  has  recently  received  much 


248  SUSCEPTIBILITY   AND   IMMUNITY. 

attention  and  has  been  studied  especially  by  Ali-Cohen,  Massart  and 
Bordet,  Gabritchevski,  and  others. 

According  to  Gabritchevski,  the  following  substances  have  a  neg- 
ative chemiotaxis  for  the  leucocytes  :  Sodium  chloride  in  ten-per-cent 
solution,  alcohol  in  ten-per-cent  solution,  quinine,  lactic  acid,  gly- 
cerin, chloroform,  bile.  On  the  other  hand,  a  positive  chemiotaxis 
is  excited  by  sterilized  or  non-sterilized  cultures  of  various  bacteria. 
This  is  shown  by  the  fact  that  when  a  small  capillary  tube,  closed  at 
one  end,  which  contains  the  substance  to  be  tested,  is  introduced  be- 
neath the  skin  of  an  animal,  the  leucocytes  are  repelled  from  the  tube 
by  certain  substances,  while  those  which  incite  positive  chemiotaxis 
cause  them  to  enter  the  tube  in  great  numbers.  The  experiments  of 
Buchner  seem  to  show  that  the  positive  chemiotaxis  induced  by 
sterilized  cultures  of  bacteria  introduced  beneath  the  skin  of  an 
animal,  is  due  to  the  proteid  contents  of  the  cells  rather  than  to  the 
chemical  products  elaborated  as  a  result  of  their  vital  activity.  But 
that  such  chemical  products  may,  in  some  instances  at  least,  produce 
a  positive  chemiotaxis  independently  of  the  bacteria  is  shown  by 
the  experiments  of  Gabritchevski  with  filtered  cultures  of  Bacillus 
pyocyaneus — confirmed  by  Massart  and  Bordet. 

An  important  observation  made  by  Bouchard,  and  confirmed  by 
Massart  and  Bordet,  is  the  following :  When  a  tube  containing  a  cul- 
ture of  Bacillus  pyocyaneus  is  introduced  beneath  the  skin  of  a  rabbit 
it  is  found,  at  the  end  of  a  few  hours,  to  contain  a  great  number  of 
leucocytes.  But  if  immediately  after  its  introduction  ten  cubic  centi- 
metres of  a  sterilized  culture  of  the  same  bacillus  are  injected  into  the 
circulation  through  a  vein,  very  few  leucocytes  enter  the  tube  intro- 
duced beneath  the  skin — that  is,  the  chemiotaxis  of  the  leucocytes 
for  the  bacilli  contained  in  the  tube  has  been  neutralized  by  injecting 
a  considerable  quantity  of  the  soluble  products  of  the  same  bacillus 
into  the  circulation. 

Buchner,  having  shown  that  the  bacterial  cells  contain  a  proteid 
substance  which  attracts  the  leucocytes,  experimented  with  various 
other  proteids  and  found  that  gluten,  casein  from  wheat,  and  legumin 
from  peas  had  a  similar  effect.  Starch  has  no  effect,  but  a  mass  of 
flour,  made  from  wheat  or  from  peas,  introduced  beneath  the  skin  of 
a  rabbit  or  of  a  guinea-pig,  with  antiseptic  precautions,  in  the  course 
of  a  day  or  two  is  enveloped  and  penetrated  by  immense  numbers  of 
leucocytes.  If,  instead  of  introducing  these  substances  which  induce 
positive  chemiotaxis  beneath  the  skin,  they  are  injected  into  the  cir- 
culation, Buchner  has  shown  that  a  great  increase  in  the  number  of 
leucocytes  occurs. 


SUSCEPTIBILITY   AND    IMMUNITY. 
THEORIES   OF   IMMUNITY. 

Exhaustion  Theory. — For  a  time  Pasteur  supported  the  view 
that  during  an  attack  of  an  infectious  disease  the  pathogenic  micro- 
organism, in  its  multiplication  in  the  body  of  a  susceptible  animal, 
exhausts  the  supply  of  some  substance  necessary  for  its  development, 
that  this  substance  is  not  subsequently  reproduced,  and  that  conse- 
quently the  same  pathogenic  germ  cannot  again  multiply  in  the  body 
of  the  protected  animal.  This  view  is  sustained  in  a  memoir  pub- 
lished in  the  Comptes  Rendus  of  the  French  Academy  in  1880,  in 
which  Pasteur  says  : 

"  It  is  the  life  of  a  parasite  in  the  interior  of  the  body  which  produces  the 
malady  commonly  called  ' cholera  des  ponies,'  and  which  causes  death. 
From  the  moment  when  this  culture  (i.e.,  the  multiplication  of  the  parasite) 
is  110  longer  possible  in  the  fowl  the  sickness  cannot  appear.  The  fowls  are 
then  in  the  constitutional  state  of  fowls  not  subject  to  be  attacked  by  the 
disease.  These  last  are  as  if  vaccinated  from  birth  for  this  malady,  because 
the  foetal  evolution  has  not  introduced  into  their  bodies  the  material  neces- 
sary to  support  the  life  of  the  microbe,  or  these  nutritive  materials  have 
disappeared  at  an  early  age. 

"Certainly  one  should  not  be  surprised  that  there  may  be  constitutions 
sometimes  susceptible  and  sometimes  rebellious  to  inoculation — that  is  to 
say,  to  the  cultivation  of  a  certain  virus — when,  as  I  have  announced  in  my 
first  note,  one  sees  a  preparation  of  beer  yeast  made,  exactly  like  one  from 
the  muscles  of  fowls  (bouillon),  to  show  itself  absolutely  unsuited  for  the  cul- 
tivation of  the  parasite  of  fowl  cholera,  while  it  is  admirably  adapted  to  the 
cultivation  of  a  multitude  of  microscopic  species,  notably  to  the  bacteride 
charbonneuse  (Bacillus  aiithracis). 

"The  explanation  to  which  these  facts  conduct  us,  as  well  of  the  consti- 
tutional resistance  of  some  individuals  as  of  the  immunity  produced  by 
protective  inoculations,  is  only  natural  when  we  consider  that  every  culture, 
in  general,  modifies  the  medium  in  which  it  is  effected — a  modification  of 
the  soil  when  it  relates  to  ordinary  plants ;  a  modification  of  plants  and  ani- 
mals when  it  relates  to  their  parasites  ;  a  modification  of  our  culture  liquids 
when  it  relates  to  mucedines,  vibrioniens,  or  ferments. 

"These  modifications  are  manifested  and  characterized  by  the  circum- 
stance that  new  cultivations  of  the  same  species  in  these  media  become 
promptly  difficult  or  impossible.  If  we  sow  chicken  bouillon  with  the  mi- 
crobe of  fowl  cholera,  and,  after  three  or  four  days,  filter  the  liquid  in  order 
to  remove  all  trace  of  the  microbe,  and  subsequently  sow  anew  in  the  fil- 
tered liquid  this  parasite,  it  will  be  found  quite  powerless  to  resume  th^  most 
feeble  development.  The  liquid,  which  is  perfectly  limpid  after  being  fil- 
tered, retains  its  limpidity  indefinitely. 

"How  can  we  fail  to  believe  that  by  cultivation  in  the  fowl  of  the  atten- 
uated virus  we  place  its  body  in  the  state  of  this  filtered  liquid  which  can 
110  longer  cultivate  the  microbe  ?  The  comparison  can  be  pushed  still 
further;  for  if  we  filter  the  bouillon  containing  the  microbe  in  full  develop- 
ment, not  on  the  fourth  day  of  culture,  but  on  the  second,  the  filtered  liquid 
will  still  be  able  to  support  the  development  of  the  microbe,  although  with 
less  energy  than  at  the  outset.  We  comprehend,  then,  that  after  a  cultiva- 
tion of  the  modified  (attenue)  microbe  in  the  body  of  the  fowl  we  may  not 
have  removed  from  all  parts  of  its  body  the  aliment  of  the  microbe.  That 
which  remains  will  permit,  then,  a  new  culture,  but  in  a  more  restricted 
measure. 

"This  is  the  effect  of  a  first  inoculation  ;  subsequent  inoculations  will 


250  SUSCEPTIBILITY   AND   IMMUNITY. 

remove  progressively  all  the  material  necessary  for  the  development  of  the 
parasite." 

In  discussing  this  theory,  in  a  paper  published  in  the  American 
Journal  of  the  Medical  Sciences  (April,  1881),  the  writer  says: 

"Let  us  see  where  this  hypothesis  leads  us.  In  the  first  place,  we  must 
have  a  material  of  small-pox,  and  a  material  of  measles,  and  a  material  of 
scarlet  fever,  etc.,  etc.  Then  we  must  admit  that  each  of  these  different 
materials  has  been  formed  in  the  system  and  stored  up  for  these  emergencies 
—attacks  of  the  diseases  in  question — for  we  can  scarcely  conceive  that  they 
were  all  packed  away  in  the  germ  cell  of  the  mother  and  the  sperm  cell  of 
the  father  of  each  susceptible  individual.  If,  then,  these  peculiar  materials 
have  been  formed  and  stored  up  during  the  development  of  the  individual, 
how  are  we  to  account  for  the  fact  that  no  new  production  takes  place  after 
an  attack  of  any  one  of  the  diseases  in  question  ? 

"  Again,  how  shall  we  account  for  the  fact  that  the  amount  of  material 
which  would  nourish  the  small-pox  germ,  to  the  extent  of  producing  a  case 
of  confluent  small-pox,  may  be  exhausted  by  the  action  of  the  attenuated 
virus  (germ)  introduced  by  vaccination  ?  Pasteur's  comparison  of  a  fowl 
protected  by  inoculation  with  the  microbe  of  fowl  cholera,  with  a  culture 
fluid  in  which  the  growth  of  a  particular  organism  has  exhausted  the  pabu- 
lum necessary  for  the  development  of  additional  organisms  of  the  same  kind, 
does  not  seem  to  me  to  be  a  just  one,  as  in  the  latter  case  we  have  a  limited 
supply  of  nutriment,  while  in  the  former  we  have  new  supplies  constantly 
provided  of  the  material — food — from  which  the  whole  body,  including  the 
hypothetical  substance  essential  to  the  development  of  the  disease  germ,  was 
built  up  prior  to  the  attack.  Besides  this  we  have  a  constant  provision  for 
the  elimination  of  effete  and  useless  products. 

"  This  hypothesis,  then,  requires  the  formation  in  the  human  body,  and 
the  retention  up  to  a  certain  time,  of  a  variety  of  materials  which,  so  far  as 
we  can  see,  serve  no  purpose  except  to  nourish  the  germs  of  various  specific 
diseases,  and  which,  having  served  this  purpose,  are  not  again  formed  in  the 
same  system,  subjected  to  similar  external  conditions,  and  supplied  with  the 
same  kind  of  nutriment." 

It  is  unnecessary  to  discuss  this  hypothesis  any  further,  inasmuch 
as  it  is  no  longer  sustained  by  Pasteur  or  his  pupils,  and  is  evidently 
untenable. 

The  Eetention  Theory,  proposed  by  Chauveau  (1880),  is  subject  to 
similar  objections.  According  to  this  view,  certain  products  formed 
during  the  development  of  a  pathogenic  microorganism  in  the  body 
of  a  susceptible  animal  accumulate  during  the  attack  and  are  subse- 
quently retained,  and,  being  prejudicial  to  the  growth  of  the  particu- 
lar microorganism  which  produced  them,  a  second  infection  cannot 
occur.  Support  for  this  theory  has  been  found  by  its  advocates  in 
the  fact  that  various  processes  of  fermentation  are  arrested  after  a 
time  by  the  formation  of  substances  which  restrain  the  development 
of  the  microorganisms  to  which  they  are  due.  But  in  the  case  of  a 
living  animal  the  conditions  are  very  different,  and  it  is  hard  to  con- 
ceive that  adventitious  products  of  this  kind  could  be  retained  for 
years,  when  in  the  normal  processes  of  nutrition  and  excretion  the 
tissues  and  fluids  of  the  body  are  constantly  undergoing  change. 
Certainly  the  substances  which  arrest  ordinary  processes  of  fermen- 


SUSCEPTIBILITY   AXD    IMMUNITY.  251 

tation  by  their  accumulation  in  the  fermenting  liquid,  such  as  alco- 
hol, lactic  acid,  phenol,  etc.,  would  not  be  so  retained.  But  we  can- 
not speak  so  positively  with  reference  to  the  toxic  albuminous 
substances  which  recent  researches  have  demonstrated  to  be  present 
in  cultures  of  some  of  the  best  known  pathogenic  bacteria.  It  is 
difficult,  however,  to  believe  that  an  individual  who  has  passed 
through  attacks  of  half  a  dozen  different  infectious  diseases  carries 
about  Avith  him  a  store  of  as  many  different  chemical  substances  pro- 
duced during  these  attacks,  and  sufficient  in  quantity  to  prevent  the 
development  of  the  several  germs  of  these  diseases.  Nor  does  the 
experimental  evidence  relating  to  the  action  of  germicide  and  germ- 
restraining  agents  justify  the  view  that  a  substance  capable  of 
preventing  the  development  of  one  microorganism  should  be  with- 
out effect  upon  others  of  the  same  class ;  but  if  we  accept  the  re- 
tention hypothesis  we  must  admit  that  the  inhibiting  substance 
produced  by  each  particular  pathogenic  germ  is  effective  only  in 
restraining  the  development  of  the  microbe  which  produced  it  in  the 
first  instance. 

Pasteur  discusses  this  hypothesis  in  his  paper  from  which  we 
have  already  quoted,  as  follows  : 

4 '  We  may  admit  the  possibility  that  the  development  of  the  microbe,  in 
place  of  removing1  or  destroying-  certain  matters  in  the  bodies  of  the  fowls, 
adds,  on  the  contrary,  something  which  is  an  obstacle  to  the  future  develop- 
ment of  this  microbe.  The  history  of  the  life  of  inferior  beings  authorizes 
such  a  supposition.  The  excretions  resulting  from  vital  processes  may  arrest 
vital  processes  of  the  same  nature.  In  certain  fermentations  we  see  anti- 
septic products  make  their  appearance  during,  and  as  a  result  of,  the  fer- 
mentation, which  put  an  end  to  the  active  life  of  the  ferments  and  arrest 
the  fermentations  long  before  they  are  completed.  In  the  cultivation  of  our 
microbe,  products  may  have  been  formed  the  presence  of  which,  possibly, 
may  explain  the  protection  following  inoculation. 

"Our  artificial  cultures  permit  us  to  test  the  truth  of  this  hypothesis. 
Let  us  prepare  an  artificial  culture  of  the  microbe,  and  after  having  evapo- 
rated it,  in  vacuo,  without  heat,  let  us  bring  it  back  to  its  original  volume 
by  means  of  fresh  chicken  bouillon.  If  the  extract  contains  a  poison  for 
the  life  of  the  microbe,  and  if  this  is  the  cause  of  its  failure  to  multiply  in  the 
fi  Itered  liquid,  the  new  liquid  should  remain  sterile.  Now,  this  is  not  the  case. 
We  cannot,  then,  believe  that  during  the  life  of  the  parasite  certain  substances 
are  produced  which  are  capable  of  arresting  its  ulterior  development." 

This  experiment  of  Pasteur  appears  to  be  conclusive  so  far  as  the 
particular  pathogenic  microorganism  referred  to  is  concerned ;  and 
we  may  say,  in  brief,  that  more  recent  investigations  do  not  sustain 
the  view  that  acquired  immunity  is  due  to  the  retention  of  products 
such  as  are  formed  by  pathogenic  bacteria  in  artificial  culture  media, 
and  which  act  by  destroying  these  bacteria  or  restraining  their  devel- 
opment when  they  are  introduced  into  the  bodies  of  immune  animals. 

Moreover,  if  we  suppose  that  the  toxic  substances  which  give 
pathogenic  power  to  a  particular  microorganism  are  retained  in  the 


252  SUSCEPTIBILITY   AND    IMMUNITY. 

body  of  an  immune  animal,  we  must  admit  that  the  animal  has  ac- 
quired a  tolerance  to  the  pathogenic  action  of  these  toxic  substances, 
for  their  presence  no  longer  gives  rise  to  any  morbid  phenomena. 
And  this  being  the  case,  we  are  not  restricted  to  the  explanation 
that  immunity  depends  upon  a  restraining  influence  exercised  upon 
the  microbe  when  subsequently  introduced. 

The  Vital  Resistance  Theory. — Another  explanation  offers  itself, 
viz.,  that  immunity  depends  upon  an  acquired  tolerance  to  the 
toxic  products  of  pathogenic  bacteria.  This  is  a  view  which  the 
writer  has  advocated  in  various  published  papers  since  1881.  In  a 
paper  contributed  to  the  American  Journal  of  the  Medical  Sci- 
ences in  April,  1881,  it  is  presented  in  the  following  language: 

"The  view  that  I  am  endeavoring-  to  elucidate  is  that,  during  a  non- 
fatal  attack  of  one  of  the  specific  diseases,  the  cellular  elements  implicated 
which  do  not  succumb  to  the  destructive  influence  of  the  poison  acquire  a 
tolerance  to  this  poison  which  is  transmissible  to  their  progeny,  and  which 
is  the  reason  of  the  exemption  which  the  individual  enjoys  from  future 
attacks  of  the  same  disease." l 

In  my  chapter  on  "Bacteria  in  Infectious  Diseases,"  in  "Bac- 
teria," published  in  the  spring  of  1884,  but  placed  in  the  hands  of  the 
publishers  in  1883,  I  say: 

"  It  may  be  that  the  true  explanation  of  the  immunity  afforded  by  a  mild 
attack  of  an  infectious  germ  disease  is  to  be  found  in  an  acquired  tolerance  to 
the  action  of  a  chemical  poison  produced  by  the  microorganism,  and  conse- 
quent ability  to  bring  the  resources  of  nature  to  bear  to  restrict  invasion  by 
the  parasite. " 

The  "resources  of  nature"  are  referred  to  in  the  same  chapter  as 
follows  : 

' '  The  hypothesis  of  Pasteur  would  account  for  the  fact  that  one  individual 
suffers  a  severe  attack  and  another  a  mild  attack  of  an  infectious  disease, 
after  being  subjected  to  the  influence  of  the  poison  under  identical  circum- 
stances, by  the  supposition  that  the  pabulum  required  for  the  development 
of  this  particular  poison  is  more  abundant  in  the  body  of  one  individual 
than  in  the  other.  The  explanation  which  seems  to  us  more  satisfactory  is 
that  the  vital  resistance  offered  by  the  cellular  elements  in  the  bodies  of 
these  two  individuals  was  not  the  same  for  this  poison.  It  is  well  known 
that  in  conditions  of  lowered  vitality  resulting  from  starvation,  profuse 
discharges,  or  any  other  cause,  the  power  to  resist  disease  poisons  is  greatly 
diminished,  and,  consequently,  that  the  susceptibility  of  the  same  individual 
differs  at  different  times. 

"From  our  point  of  view,  the  blood,  as  it  is  found  within  the  vessels  of  a 
living  animal,  is  not  simply  a  culture  fluid  maintained  at  a  fixed  tempera- 
ture, but  under  these  circumstances  is  a  tissue,  the  histological  elements  of 
which  present  a  certain  vital  resistance  to  pathogenic  organisms  which  may 
be  introduced  into  the  circulation. 

"  If  we  add  a  small  quantity  of  a  culture  fluid  containing  the  bacteria  of 
putrefaction  to  the  blood  of  an  animal,  withdrawn  from  the  circulation  into 
a  proper  receptacle  and  maintained  in  a  culture  oven  at  blood  heat,  we  will 
find  that  these  bacteria  multiply  abundantly,  and  evidence  of  putrefactive 

1  "  "What  is  the  Explanation  of  the  Protection  from  Subsequent  Attacks,  result- 
ing from  an  Attack  of  Certain  Diseases,  etc  ? ''  American  Journal  of  the  Medical 
Sciences,  April,  1881,  p.  37«. 


SUSCEPTIBILITY   AXD    IMMUNITY.  263 

decomposition  will  soon  be  perceived.  But  if  we  inject  a  like  quantity  of 
the  culture  fluid  with  its  contained  bacteria  into  the  circulation  of  a  living 
animal,  not  only  does  no  increase  and  110  putrefactive  change  occur,  but  the 
bacteria  introduced  quickly  disappear,  and  at  the  end  of  an  hour  or  two  the 
most  careful  microscopical  examination  will  not  reveal  the  presence  of  a 
single  bacterium.  This  difference  we  ascribe  to  the  vital  properties  of  the 
fluid  as  contained  in  the  vessels  of  a  living  animal;  and  it  seems  probable 
that  the  little  masses  of  protoplasm  known  as  white  blood  corpuscles  are  the 
essential  histological  elements  of  the  blood,  so  far  as  any  manifestation  of 
vitality  is  concerned.  The  writer  has  elsewhere  (1881)  suggested  that  the 
disappearance  of  the  bacteria  from  the  circulation,  in  the  experiment 
referred  to,  may  be  effected  by  the  white  corpuscles,  wrhich,  it  is  well  known, 
pick  up,  after  the  manner  of  amoebae,  any  particles,  organic  or  inorganic, 
which  come  in  their  way.  And  it  requires  no  great  stretch  of  credulity  to 
believe  that  they  may,  like  an  amoeba,  digest  and  assimilate  the  protoplasm 
of  the  captured  bacterium,  thus  putting  an  end  to  the  possibility  of  its  do- 
ing any  harm. 

"  In  the  case  of  a  pathogenic  organism  we  may  imagine  that,  when  cap- 
tured in  this  way,  it  may  share  a  like  fate  if  the  captor  is  not  paralyzed  by 
some  potent  poison  evolved  by  it,  or  overwhelmed  by  its  superior  vigor  and 
rapid  multiplication.  In  the  latter  event  the  active  career  of  our  conserva- 
tive white  corpuscle  would  be  quickly  terminated  and  its  protoplasm  would 
serve  as  food  for  the  enemy.  It  is  evident  that  in  a  contest  of  this  kind  the 
balance  of  power  would  depend  upon  circumstances  relating  to  the  inherited 
vital  characteristics  of  the  invading  parasite  and  of  the  invaded  leucocyte." 

In  the  same  chapter  the  writer  quotes  from  his  paper  on  acquired 
immunity,  published  in  1881,  as  follows  : 

' '  The  difficulties  into  which  this  hypothesis  [the  exhaustion  theory  of  Pas- 
teur] leads  us  certainly  justify  us  in  looking  further  for  an  explanation  of  the 
phenomena  in  question.  This  explanation  is,  I  believe,  to  be  found  in  the 
peculiar  properties  of  the  protoplasm,  which  is  the  essential  framework  of 
every  living  organism.  The  properties  referred  to  are  the  tolerance  which 
living  protoplasm  may  acquire  to  certain  agents  which,  in  the  first  instance, 
have  an  injurious  or  even  fatal  influence  upon  its  vital  activity  ;  and  the 
property  which  it  possesses  of  transmitting  its  peculiar  qualities,  inherent  or 
acquired,  through  numerous  generations,  to  its  offshoots  or  progeny. 

tk  Protoplasm  is  the  essential  living  portion  of  the  cellular  elements  of  ani- 
mal and  vegetable  tissues ;  but  as  our  microscopical  analysis  of  the  tissues  has 
not  gone  beyond  the  cells  of  which  they  are  composed,  and  is  not  likely  to 
reveal  to  us  the  complicated  molecular  structure  of  the  protoplasm,  upon 
which,  possibly,  the  properties  under  consideration  depend,  it  will  be  best, 
for  the  present,  to  limit  ourselves  to  a  consideration  of  the  living  cells  of  the 
body.  These  cells  are  the  direct  descendants  of  the  pre-existent  cells,  and 
may  all  be  traced  back  to  the  sperm  cell  and  the  germ  cell  of  the  parents. 
Now,  the  view  which  I  am  endeavoring  to  elucidate  is  that,  during  a  non- 
fatal  attack  of  one  of  the  specific  diseases,  the  cellular  elements  implicated, 
which  do  not  succumb  to  the  destructive  influence  of  the  poison,  acquire  a 
tolerance  to  this  poison  which  is  transmissible  to  their  progeny,  and  which 
is  the  reason  of  the  exemption  which  the  individual  enjoys  from  future 
attacks  of  the  same  disease. 

"  The  known  facts  in  regard  to  the  hereditary  transmission  by  cells  of  ac- 
quired properties'  make  it  easy  to  believe  in  the  transmission  of  such  a 
tolerance  as  we  imagine  to  be  acquired  during  the  attack ;  and  if  it  is  shown 
by  analogy  that  there  is  nothing  improbable  in  the  hypothesis  that  such  a 
tolerance  is  acquired,  we  shall  have  a  rational  explanation,  not  of  heredity 
and  of  the  mysterious  properties  of  protoplasm,  but  of  the  particular  result 
under  consideration.  The  transmission  of  acquired  properties  is  shown  in 
the  budding  and  grafting  of  choice  fruits  and  flowers,  produced  by  cultiva- 


254  SUSCEPTIBILITY   AND    IMMUNITY, 

tion,  upon  the  wild  stock  from  which  they  originated.  The  acquired  proper- 
ties are  transmitted  indefinitely;  and  the  same  sap  which  on  one  twig-  nour- 
ishes a  sour  crab  apple,  on  another  one  of  the  same  branch  is  elaborated  into 
a  delicious  pippin. 

'  The  tolerance  to  narcotics— opium  and  tobacco — and  to  corrosive  poisons 
— arsenic — which  results  from  a  gradual  increase  of  dose,  may  be  cited  as  an 
example  of  acquired  tolerance  by  living  protoplasm  to  poisons  which  at  the 
outset  would  have  been  fatal  in  much  smaller  doses. 

'The  immunity  which  an  individual  enjoys  from  any  particular  disease 
must  be  looked  upon  as  a  power  of  resistance" possessed  by  the  cellular  ele- 
ments of  those  tissues  of  his  body  which  would  yield  to  the  poison  in  the 
case  of  an  unprotected  person." 

This  theory  of  immunity,  advanced  by  the  author  in  1881,  has 
received  considerable  support  from  investigations  made  since  that 
date,  and  especially  from  the  experimental  demonstration  by  Sal- 
mon, Roux,  and  others  that,  as  suggested  in  the  paper  from  which  I 
have  quoted,  immunity  may  result  from  the  introduction  into  the 
body  of  a  susceptible  animal  of  the  soluble  products  of  bacterial 
growth — filtered  cultures. 

The  theory  of  vital  resistance  to  the  toxic  products  evolved  by 
pathogenic  bacteria  is  also  supported  by  numerous  experiments 
which  show  that  natural  or  acquired  immunity  may  be  overcome 
when  these  toxic  products  are  introduced  in  excess,  or  when  the  vital 
resisting  power  of  the  animal  has  been  reduced  by  various  agencies. 

More  direct  experimental  evidence  in  favor  of  the  view  under  con- 
sideration is  that  obtained  by  Beumer  in  his  experiments  with  steril- 
ized cultures  of  the  typhoid  bacillus.  He  found  that  after  the  re- 
peated injection  of  non-lethal  doses  mice  were  able  to  resist  an 
amount  of  this  toxine  which  was  fatal  to  animals  of  the  same  spe- 
cies not  so  treated.  But,  on  the  other  hand,  Gamaleia  found,  in  his 
experiments  upon  guinea-pigs  which  had  been  made  immune  against 
the  pathogenic  action  of  a  spirillum,  called  by  him  Vibrio  Metschni- 
kovi,  that  these  animals  have  no  increased  tolerance  for  the  toxic 
products  of  this  microorganism.  Although  immune  against  infec- 
tion by  the  living  microbe,  they  were  killed  by  the  same  quantity  of 
a  sterilized  culture  as  was  fatal  to  guinea-pigs  which  had  not  been 
rendered  immune. 

Charrin  has  obtained  similar  results  in  experiments  with  filtered 
cultures  of  Bacillus  pyocyaneus.  Rabbits  which  had  an  artificial  im- 
munity against  the  pathogenic  action  of  the  bacillus  were  killed  by 
doses  of  a  sterilized  culture  such  as  were  fatal  to  other  rahbits  of  the 
same  size  not  immune.  In  subsequent  experiments  by  Charrin  and 
Gameleia  "  vaccinated  "  rabbits  were  found  to  be  even  more  suscepti- 
ble to  the  toxic  action  of  filtered  cultures  than  were  those  not  vacci- 
nated. Metschnikoff  (1891)  has  followed  up  this  line  of  experiment, 
and  has  shown  that  when  considerable  amounts  of  filtered  cultures 
of  Bacillus  pyocyaneus  are  injected  subcutaneously  in  rabbits  a  cer- 


SUSCEPTIBILITY   AND   IMMUNITY.  255 

tain  tolerance  to  the  toxic  action  of  the  same  cultures  is  established 
in  some  instances.  But  his  results  do  not  give  any  substantial  sup- 
port to  the  view  that  immunity  depends  upon  an  acquired  tolerance 
to  the  toxic  action  of  the  chemical  products  contained  in  cultures  of 
the  pathogenic  bacteria  with  which  he  experimented— Bacillus  pyo- 
cyaneus  and  Vibrio  Metschnikovi. 

In  view  of  the  results  of  experimental  researches  above  recorded, 
and  of  other  recent  experiments  which  show  that,  in  certain  cases  at 
least,  acquired  immunity  depends  upon  the  formation  of  an  anti- 
toxine  in  the  body  of  the  immune  animal,  we  are  convinced  that  the 
theory  of  immunity  under  discussion,  first  proposed  by  the  writer  in 
1881,  cannot  be  accepted  as  a  sufficient  explanation  of  the  facts  in 
general.  At  the  same  time  we  are  inclined  to  attribute  considerable 
importance  to  acquired  tolerance  to  the  toxic  products  of  pathogenic 
bacteria  as  one  of  the  factors  by  which  recovery  from  an  infectious 
disease  is  made  possible  and  subsequent  immunity  established. 

The  "  vital-resistance  theory"  of  the  present  writer,  as  set  forth, 
in  the  above-quoted  extracts  from  his  published  papers,  is  essentially 
the  same  as  that  advocated  by  Buchner  at  a  later  date  (1883).  Buch- 
ner  supposes  that  during  the  primary  infection,  when  an  animal  re- 
covers, a  "  reactive  change  "  has  been  produced  in  the  cells  of  the 
body  which  enables  it  to  protect  itself  from  the  pathogenic  action 
of  the  same  microorganism  when  subsequently  introduced. 

Of  course  when  we  ascribe  immunity  to  the  "  vital  resistance"  of 
the  cellular  elements  of  the  body,  we  have  not  explained  the 
modus  operandi  of  this  vital  resistance  or  "  reactive  change,"  but 
have  simply  affirmed  that  the  phenomenon  in  question  depends  upon 
some  acquired  property  residing  in  the  living  cellular  elements  of 
the  body.  We  have  suggested  that  that  which  has  been  acquired 
is  a  tolerance  to  the  action  of  the  toxic  products  produced  by  patho- 
genic bacteria.  But,  as  already  stated,  in  the  light  of  recent  experi- 
ments this  theory  now  appears  to  us  to  be  untenable  as  a  general 
explanation  of  acquired  immunity. 

The  Theory  of  Phagocytosis. — The  fact  that  in  certain  infectious 
diseases  due  to  bacteria  the  parasitic  invaders,  at  the  point  of  inocu- 
lation or  in  the  general  blood  current,  are  picked  up  by  the  leuco- 
cytes and  in  properly  stained  preparations  may  be  seen  in  their  in- 
terior, has  been  known  for  some  years.  In  mouse  septica3mia — an 
infectious  disease  described  by  Koch  in  his  work  on  "Traumatic 
Infectious  Diseases,"  published  in  1878 — the  slender  bacilli  which  are 
the  cause  of  the  disease  are  found  in  large  numbers  in  the  interior  of 
the  leucocytes.  Koch  says,  in  the  work  referred  to  :  "  Their  rela- 
tion to  the  white  blood  corpuscles  is  peculiar  ;  they  penetrate  these 
and  multiply  in  their  interior.  One  often  finds  that  there  is 


256  SUSCEPTIBILITY   AND    IMMUNITY. 

hardly  a  single  white  corpuscle  in  the  interior  of  which  bacilli  can- 
not be  seen.  Many  corpuscles  contain  isolated  bacilli  only;  others 
have  thick  masses  in  their  interior,  the  nucleus  being  still  recog- 
nizable ;  while  in  others  the  nucleus  can  be  no  longer  distinguished  ; 
and,  finally,  the  corpuscle  may  become  a  cluster  of  bacilli,  breaking 
up  at  the  margin — the  origin  of  which  one  could  not  have  explained 
had  there  been  no  opportunity  of  seeing  all  the  intermediate  steps 
between  the  intact  white  corpuscle  and  these  masses"  (Fig.  78).  It 
will  be  noted  that  in  the  above  quotation  Koch  affirms  that  the 
bacilli  penetrate  the  leucocytes  and  multiply  in  their  interior.  Now, 
the  theory  of  phagocytosis  assumes  that  the  bacilli  are  picked  up  by 
the  leucocytes  and  destroyed  in  their  interior,  and  that  immunity  de- 
pends largely  upon  the  power  of  these  "phagocytes"  to  capture  and 
destroy  living  pathogenic  bacilli. 

The  writer  suggested  this  as  an  hypothesis  as  long  ago  as  1881, 
in  a  paper  read  before  the  American  Association  for  the  Advance- 
ment of  Science,  in  the  following  language: 

"  It  has  occurred  to  me  that  possibly  the  white  corpuscles  may 
have  the  office  of  picking  up  and  digesting  bacterial  organisms  which 


FIG.  78.— Bacillus  of  mouse  septicaemia  in  leucocytes  from  blood  of  mouse  (Koch). 

by  any  means  find  their  way  into  the  blood.  The  propensity  exhib- 
ited by  the  leucocytes  for  picking  up  inorganic  granules  is  well 
known,  and  that  they  may  be  able  not  only  to  pick  up  but  to  assimi- 
late, and  so  dispose  of,  the  bacteria  which  come  in  their  way,  does 
not  seern  to  me  very  improbable,  in  view  of  the  fact  that  amcebaB, 
which  resemble  them  so  closely,  feed  upon  bacteria  and  similar  or- 
ganisms." 

At  a  later  date  (1884)  Metschnikoff  offered  experimental  evi- 
dence in  favor  of  this  view,  and  the  explanation  suggested  in  the 
above  quotation  is  commonly  spoken  of  as  the  Metschnikoff  theory. 

1  "  A  Contribution  to  the  Study  of  Bacterial  Organisms  commonly  found  upon 
Exposed  Mucous  Surf  aces  and  in  the  Alimentary  Canal  of  Healthy  Individuals."  Il- 
lustrated by  photomicrographs.  Proceedings  of  the  American  Association  for  Ad- 
vancement of  Science,  1881,  Salem,  1882,  xxx.,  83-94.  Also  in  Studies  from  the 
Biological  Laboratory,  Johns  Hopkins  University,  Baltimore,  vol.  ii.,  No.  2,  1882. 


SUSCEPTIBILITY    AND    IMMUNITY.  257 

The  observations  which  first  led  Metschnikoff  to  adopt  this  view 
were  made  upon  a  species  of  daphnia  which  is  subject  to  fatal  infec- 
tion by  a  torula  resembling  the  yeast  fungus.  Entering  with  the 
food,  this  fungus -penetrates  the  walls  of  the  intestine  and  invades  the 
tissues.  In  certain  cases  the  infection  does  not  prove  fatal,  owing,  as 
Metschnikoff  asserts,  to  the  fact  that  the  fungus  cells  are  seized  upon 
by  the  leucocytes,  which  appear  to  accumulate  around  the  invading 
parasite  (chemiotaxis)  for  this  special  purpose.  If  they  are  success- 
ful in  overpowering  and  destroying  the  parasite  the  animal  recovers  ; 
if  not,  it  succumbs  to  the  general  infection  which  results.  In  a  simi- 
lar manner,  Metschnikoff  supposes,  pathogenic  bacteria  are  destroyed 
when  introduced  into  the  body  of  an  immune  animal.  The  colorless 
blood  corpuscles,  which  he  designates  phagocytes,  accumulate  at  the 
point  of  invasion  and  pick  up  the  living  bacteria,  as  they  are  known 
to  pick  up  inorganic  particles  injected  into  the  circulation.  So  far 
there  can  be  no  doubt  that  Metschnikoff  is  right.  The  presence  of 
bacteria  in  the  leucocytes  in  considerable  numbers,  'both  at  the  point 
of  inoculation  and  in  the  general  circulation,  has  been  repeatedly 
demonstrated  in  animals  inoculated  with  various  pathogenic  bacteria. 
The  writer  observed  this  in  his  experiments,  made  in  1881,  in  which 
rabbits  were  inoculated  with  cultures  of  his  Micrococcus  Pasteuri  ; 
and  it  was  this  observation  which  led  him  to  suggest  the  theory 
which  has  since  been  so  vigorously  supported  by  Metschnikoff.  But 
the  presence  of  a  certain  number  of  bacteria  within  the  leucocytes 
does  not  prove  the  destructive  power  of  these  cells  for  living  patho- 
genic organisms.  As  urged  by  Weigert,  Baumgarten,  and  others, 
,  it  may  be  that  the  bacteria  were  already  dead  when  they  were  picked 
up,  having  been  destroyed  by  some  agency  outside  of  the  blood  cells. 
As  heretofore  stated,  we  have  now  experimental  evidence  that  blood 
serum,  quite  independently  of  the  cellular  elements  contained  in  it 
in  the  circulation,  has  decided  germicidal  power  for  certain  patho- 
genic bacteria,  and  that  the  blood  serum  of  the  rat  and  other  animals 
which  have  a  natural  immunity  against  anthrax  is  especially  fatal 
to  the  anthrax  bacillus. 

Numerous  experiments  have  been  made  with  a  view  to  deter- 
mining whether  pathogenic  bacteria  are,  in  fact,  destroyed  within 
the  leucocytes  after  being  picked  up,  and  different  experimenters 
have  arrived  at  different  conclusions.  In  the  case  of  mouse  septi- 
csemia,  already  alluded  to,  and  in  gonorrhoea,  one  would  be  disposed 
to  decide,  from  the  appearance  and  arrangement  of  the  pathogenic 
bacteria  in  the  leucocytes,  that  they  are  not  destroyed,  but  that, 
on  the  other  hand,  they  multiply  in  the  interior  of  these  cells,  which 
in  the  end  succumb  to  this  parasitic  invasion.  In  both  of  the  dis- 
eases mentioned  we  find  leucocytes  so  completely  filled  with  the 
17 


258  SUSCEPTIBILITY   AND   IMMUNITY. 

pathogenic  microorganisms  that  it  is  difficult  to  believe  that  they 
have  all  been  picked  up  by  .  a  voracious  phagocyte,  which  has 
stuffed  itself  to  repletion,  while  numerous  other  leucocytes  from 
the  same  source  and  in  the  same  microscopic  field  of  view  have 
failed  to  capture  a  single  bacillus  or  micrococcus.  Moreover,  the 
staining  of  the  parasitic  invaders,  and  the  characteristic  arrange- 
ment of  the  "gonococcus"  in  stained  preparations  of  gonorrhceal 
pus,  indicate  that  their  vitality  has  not  been  destroyed  in  the  interior 
of  the  leucocytes  or  pus  cells,  and  we  can  scarcely  doubt  that  the 
large  number  found  in  certain  cells  is  due  to  multiplication  in  situ 
rather  than  to  an  unusual  activity  of  these  particular  cells.  But  in 
certain  infectious  diseases,  and  especially  in  anthrax,  the  bacilli  in- 
cluded within  the  leucocytes  often  give  evidence  of  degenerative 
changes,  which  would  support  the  view  that  they  are  destroyed  by 
the  leucocytes,  unless  these  changes  occurred  before  they  were  picked 
up,  as  is  maintained  by  Nuttall  and  others.  We  cannot  consider 
this  question  as  definitely  settled. 

Going  back  to  the  demonstrated  fact  that  susceptible  animals  may 
be  made  immune  by  inoculating  them  with  the  toxic  products  pro- 
duced during  the  growth  of  certain  pathogenic  bacteria,  we  may 
suppose  either  that  immunity  results  from  the  continued  presence  of 
these  toxic  products  in  the  body  of  the  inoculated  animal,  or  from  a 
tolerance  acquired  at  the  time  of  the  inoculation  and  subsequently 
retained — by  transmission  from  cell  to  cell,  as  heretofore  suggested. 
Under  the  first  hypothesis — retention  theory — immunity  may  be  ex- 
plained as  due  to  a  continued  tolerance  on  the  part  of  the  cellular  ele- 
ments of  the  body  to  the  toxic  substances  introduced  and  retained  ; 
or  to  the  effect  of  these  retained  toxic  products  in  destroying  the 
pathogenic  bacteria,  or  in  neutralizing  their  products  when  these  are 
subsequently  introduced  into  the  body  of  the  immune  animal.  We 
cannot  understand  how  toxic  substances  'introduced  in  the  first  in- 
stance can  neutralize  substances  of  the  same  kind  introduced  at  a 
later  date.  There  is  something  in  the  blood  of  the  rat  which,  accord- 
ing to  Behring,  neutralizes  the  toxic  substances  present  in  a  filtered 
culture  of  the  tetanus  bacillus ;  but  whatever  this  substance  may  be, 
it  is  evidently  different  from  the  toxic  substance  which  it  destroys, 
and  there  is  nothing  in  chemistry  to  justify  the  supposition  last 
made.  Is  it,  then,  by  destroying  the  pathogenic  microorganism 
that  these  inoculated  and  retained  toxic  products  preserve  the  animal 
from  future  infection  ?  Opposed  to  this  supposition  is  the  fact  that 
the  blood  of  an  animal  made  immune  in  this  v/ay,  when  removed 
from  the  body,  does  not  prove  to  have  increased  germicidal  power  as 
compared  with  that  of  a  susceptible  animal  of  the  same  species. 
Again,  these  same  toxic  substances  in  cultures  of  the  anthrax  bacillus, 


SUSCEPTIBILITY    AND    IMMUNITY.  259 

the  tetanus  bacillus,  the  diphtheria  bacillus,  etc.,  do  not  destroy  the 
pathogenic  germ  after  weeks  or  months  of  exposure.  And  when  we 
inoculate  a  susceptible  animal  with  a  viruleift  culture  of  one  of  these 
microorganisms,  the  toxic  substances  present  do  not  prevent  the  rapid 
development  of  the  bacillus  ;  indeed,  instead  of  proving  a  germicide, 
they  favor  its  development,  which  is  more  abundant  and  rapid  than 
when  attenuated  cultures  containing  less  of  the  toxic  material  are 
used  for  the  inoculation.  In  view  of  these  facts  we  are  unable  to 
adopt  the  view  that  acquired  immunity  results  from  the  direct  action 
of  the  products  of  bacterial  growth,  introduced  and  retained  in  the 
body  of  the  immune  animal,  upon  the  pathogenic  microorganism 
when  subsequently  introduced  or  upon  its  toxic  products. 

But  there  is  another  explanation  which,  although  it  may  appear 
a  priori  to  be  quite  improbable,  has  the  support  of  recent  experimen- 
tal evidence.  This  is  the  supposition  that  some  substance  is  formed 
in  the  body  of  the  immune  animal  which  neutralizes  the  toxic 
products  of  the  pathogenic  microorganism.  How  the  presence  of 
these  toxic  products  in  the  first  instance  brings  about  the  formation 
of  an  "antitoxin  "  by  which  they  are  neutralized  is  still  a  mystery; 
but  that  such  a  substance  is  formed  appears  to  be  proved  by  the  ex- 
periments of  Ogata,  Behring  and  Kitasato,  Tizzoni  and  Cattani,  G. 
and  F.  Klemperer,  and  others. 

Ogata  and  Jasuhara,  in  a  series  of  experiments  made  in  the  Hy- 
gienic Institute  at  Tokio  (1890),  discovered  the  important  fact  that 
the  blood  of  an  animal  immune  against  anthrax  contains  some  sub- 
stance which  neutralizes  the  toxic  products  of  the  anthrax  bacillus. 
When  cultures  were  made  in  the  blood  of  dogs,  frogs,  or  of  white 
rats,  which  animals  have  a  natural  immunity  against  anthrax,  they 
were  found  not  to  kill  mice  inoculated  with  them.  Further  experi- 
ments showed  that  mice  inoculated  with  virulent  anthrax  cultures 
did  not  succumb  to  anthrax  septicaBmia  if  they  received  at  the  same 
time  a  subcutaneous  injection  of  a  small  quantity  of  the  blood  of  an 
immune  animal.  So  small  a  dose  as  one  drop  of  frog's  blood  or  one- 
half  drop  of  dog's  blood  proved  to  be  sufficient  to  protect  a  mouse 
from  the  fatal  effect  of  an  anthrax  inoculation.  And  the  protective 
inoculation  was  effective  when  made  as  long  as  seventy-two  hours 
before  or  five  hours  after  infection  with  an  anthrax  culture.  Fur- 
ther, it  was  found  that  mice  which  had  survived  anthrax  infection  as 
a  result  of  this  treatment  were  immune  at  a  later  date  (after  several 
weeks)  when  inoculated  with  a  virulent  culture  of  the  anthrax 
bacillus. 

Behring  and  Kitasato  have  obtained  similar  results  in  their  ex- 
periments upon  tetanus  and  diphtheria,  and  have  shown  that  the 
blood  of  an  immune  animal,  added  to  virulent  cultures  before  in- 


2GO  SUSCEPTIBILITY    AND    IMMUNITY. 

oculation  into  susceptible  animals,  neutralizes  the  pathogenic  power 
of  these  cultures. 

They  have  shown  by  experiment  that  the  blood  of  a  rabbit  which 
has  an  acquired  immunity  against  tetanus,  mixed  with  the  virulent 
filtrate  from  a  culture  of  the  tetanus  bacillus,  neutralizes  its  toxic 
power.  One  cubic  centimetre  of  this  filtrate  was  mixed  with  five 
cubic  centimetres  of  serum  from  the  blood  of  an  immune  rabbit  and 
allowed  to  stand  for  twenty-four  hours  ;  0. 2  cubic  centimetre  of  this 
injected  into  a  mouse  was  without  effect,  while  0.0001  cubic  centi- 
metre of  the  filtrate  without  such  admixture  was  infallibly  fatal  to 
mice.  The  mice  inoculated  with  this  mixture  remained  immune  for 
forty  to  fifty  days,  after  which  they  gradually  lost  their  immunity. 
The  blood  or  serum  from  an  immune  rabbit,  when  preserved  in  a 
dark,  cool  place,  retained  its  power  of  neutralizing  the  tetanus  tox- 
albumin  for  about  a  week,  after  which  time  it  gradually  lost  this 
power.  The  blood  of  chickens,  which  have  a  natural  immunity 
against  tetanus,  was  found  not  to  have  a  similar  power.  Behring 
and  Kitasato  have  also  shown  that  the  serum  of  a  diphtheria-immune 
rabbit  destroys  the  potent  toxalbumin  in  diphtheria  cultures.  It 
does  not,  however,  possess  any  germicidal  power  against  the  diph- 
theria bacillus. 

Ogata,  in  1891,  reported  that  he  had  succeeded  in  isolating  from  the 
blood  of  dogs  and  of  chickens  a  substance  to  which  he  ascribes  the  nat- 
ural immunity  of  these  animals  from  certain  infectious  diseases,  and 
the  power  of  their  blood  to  protect  susceptible  animals  from  the  same 
diseases.  This  substance  is  soluble  in  water  and  in  glycerin,  but  in- 
soluble in  alcohol  or  ether,  by  which  it  is  precipitated  without  being 
destroyed.  Its  activity  is  neutralized  by  acids,  but  not  by  weak 
alkaline  solutions.  Ogata  supposes  the  substance  isolated  by  him  to 
be  the  active  agent  in  blood  serum  by  which  certain  pathogenic  bac- 
teria are  destroyed,  as  shown  by  the  experiments  of  Nuttall,  Buchner, 
and  others.  Hankin  had  previously  isolated  an  albuminoid  sub- 
stance from  the  spleen  and  blood  of  the  rat,  to  which  he  ascribed  the 
immunity  of  this  animal  from  anthrax.  This  substance,  according 
to  the  author  named,  is  a  globulin;  it  is  insoluble  in  alcohol  and  in 
distilled  water,  and  does  not  dialyze. 

Tizzoni  and  Cattani  ascribe  the  protection  of  animals  which  have 
acquired,  an  immunity  against  tetanus  to  the  presence  of  an  albumi- 
nous substance  which  they  call  the  tetanus-antitoxin.  This  they 
have  isolated  from  the  blood  of  immune  animals.  They  arrive  at 
the  conclusion  that  it  is  a  globulin,  or  a  substance  which  is  carried 
down  with  the  globulin  precipitate,  and  that  it  is  different  from  the 
globulin,  above  referred  to,  obtained  by  Hankin  from  animals  im- 
mune against  anthrax. 


SUSCEPTIBILITY   AND   IMMUNITY.  261 

G.  and  F.  Klemperer,  in  1891,  published  an  important  memoir  in 
which  they  gave  an  account  of  their  researches  relating  to  the  ques- 
tion of  immunity,  etc.,  in  animals  subject  to  the  form  of  septicaBmia 
produced  by  the  Micrococcus  pneumonia  crouposaB.  They  were  able 
to  produce  immunity  in  susceptible  animals  by  introducing  into  their 
bodies  filtered  cultures  of  this  micrococcus,  and  proved  by  experiment 
that  this  immunity  had  a  duration  of  at  least  six  months.  They 
arrived  at  the  conclusion  that  the  immunity  induced  by  injecting  fil- 
tered cultures  is  not  directly  due  to  the  toxic  substances  present  in 
these  cultures,  but  that  they  cause  the  production  in  the  tissues  of  an 
antitoxin  which  has  the  power  of  neutralizing  their  pathogenic 
action.  The  toxic  substance  present  in  cultures  of  the  "diplococcus 
of  pneumonia"  they  call  "  pneumotoxin" ;  the  substance  produced  in 
the  body  of  an  artificially  immune  animal,  by  which  this  pneumo- 
toxin is  destroyed  if  subsequently  introduced,  they  call  "  anti -pneumo- 
toxin." 

Emmerich,  in  a  communication  made  at  the  meeting  of  the  In- 
ternational Congress  for  Hygiene  and  Demography,  in  London,  re- 
ported results  which  correspond  with  those  of  G.  and  F.  Klemperer 
so  far  as  the  production  of  immunity  is  concerned,  and  also  gave  an 
account  of  experiments  made  by  Donissen  in  which  the  injection  of 
twenty  to  twenty-five  cubic  centimetres  of  blood  or  expressed  tissue 
juices,  filtered  through  porcelain,  from  an  immune  rabbit  into  an 
unprotected  rabbit,  subsequently  to  infection  with  a  bouillon  culture 
of  "diplococcus  pneumonise,"  prevented  the  development  of  fatal 
septicremia.  Even  when  the  injection  was  made  twelve  to  fifteen 
hours  after  infection,  by  inhalation,  the  animal  recovered. 

Emmerich  and  Mastraum  had  previously  reported  similar  results 
in  experiments  made  upon  mice  with  the  Bacillus  erysipelatos  suis 
(rothlauf  bacillus).  White  mice  are  very  susceptible  to  the  patho- 
genic action  of  this  bacillus.  But  mice  which,  subsequently  to  in- 
fection, were  injected  with  the  expressed  and  filtered  tissue  juices  of 
an  immune  rabbit,  recovered,  while  the  control  animals  succumbed. 
According  to  Emmerich,  the  result  in  these  experiments  was  due  to 
a  destruction  of  the  pathogenic  bacilli  in  the  bodies  of  the  infected 
animals  ;  and  the  statement  is  made  that  at  the  end  of  eight  hours 
after  the  injection  of  the  expressed  tissue  juices  all  bacilli  in  the  body 
of  the  infected  animal  were  dead.  The  same  liquid  did  not,  however, 
kill  the  bacilli  when  added  to  cultures  external  to  the  body  of  an 
animal.  The  inference,  therefore,  seems  justified  that  the  result  de- 
pends, not  upon  a  substance  present  in  the  expressed  juices  of  an 
immune  animal,  but  upon  a  substance  formed  in  the  body  of  the 
animal  into  which  these  juices  are  injected. 

We  have,  however,  an  example  of  induced  immunity  in  which 


262  SUSCEPTIBILITY   AND    IMMUNITY. 

the  result  appears  to  depend  directly  upon  the  destruction  of  the 
pathogenic  microorganism  in  the  body  of  the  immune  animal.  In 
guinea-pigs  which  have  an  acquired  immunity  against  Vibrio  Metsch- 
nikovi  the  blood  serum  has  been  proved  to  possess  decided  germicidal 
power  for  this  "vibrio,"  whereas  it  multiplies  readily  in  the  blood 
serum  of  non-immune  guinea-pigs  (Behring  and  Msseii). 

There  is  experimental  evidence  that  animals  may  acquire  an  arti- 
ficial immunity  against  the  toxic  action  of  certain  toxalbumins  from 
other  sources  than  bacterial  cultures.  Thus  Sewell  (1887)  has  shown 
that  a  certain  degree  of  tolerance  to  the  action  of  rattlesnake  venom 
may  be  established  by  inoculating  susceptible  animals  with  small 
doses  of  the  "  hemialbumose "  to  which  it  owes  its  toxic  potency. 
These  results  have  been  confirmed  by  the  more  recent  experiments  of 
Calmette  (1894)  and  of  Fraser  (1895).  In  his  paper  detailing  the 
results  of  his  experiments  the  first-named  author  says : 

"Animals  may  be  immunized  against  the  venom  of  serpents  either  by 
means  of  repeated  injections  of  doses  at  first  feeble  and  progressively  stronger, 
or  by  means  of  successive  injections  of  venom  mixed  with  certain  chemical 
substances,  among  which  I  mention  especially  chloride  of  gold  and  the  hypo- 
chlorites  of  lime  or  of  soda. 

"The  serum  of  animals  thus  treated  is  at  the  same  time  preventive,  anti- 
toxic, and  therapeutic,  exactly  as  is  that  of  animals  immunized  against 
diphtheria  or  tetanus. 

"If  we  inoculate  a  certain  number  of  rabbits,  under  the  skin  of  the 
thigh,  with  the  same  dose,  one  milligramme  of  cobra  venom  for  example, 
and  if  we  treat  all  of  these  animals  with  the  exception  of  some  for  control, 
by  subcutaneous  or  intraperitoneal  injections  of  the  serum  of  rabbits  im- 
munized against  four  milligrammes  of  the  same  venom,  all  of  the  control 
animals  not  treated  will  die  within  three  or  four  hours,  while  all  of  the 
animals  will  recover  which  receive  five  cubic  centimetres  of  the  therapeutic 
serum  within  an  hour  after  receiving  the  venom." 

In  this  connection  we  may  remark  that  there  is  some  evidence  to 
show  that  persons  who  are  repeatedly  stung  by  certain  poisonous  in- 
sects— mosquitoes,  bees — acquire  a  greater  or  less  degree  of  immu- 
nit}r  from  the  distressing  local  effects  of  their  stings. 

Ehrlich,  of  Berlin,  in  1891,  reported  his  success  in  establishing 
immunity  in  guinea-pigs  against  two  toxalbumins  of  vegetable 
origin:  one — ricin — from  the  castor-oil  bean  (Ricinus  communis), 
the  other — abrin — from  the  jequirity  bean.  The  toxic  potency 
of  ricin  is  somewhat  greater  than  that  of  abrin,  and  it  is  esti- 
mated by  Ehrlich  that  one  gramme  of  this  substance  would  suffice 
to  kill  one  and  a  half  millions  of  guinea-pigs.  When  injected  be- 
neath the  skin,  in  dilute  solution,  it  produces  intense  local  inflamma- 
tion, resulting  in  necrosis  of  the  tissues.  Mice  are  less  susceptible 
tfyan  guinea-pigs  and  are  more  easily  made  immune.  This  is  most 
readily  effected  by  giving  them  small  and  gradually  increasing  doses 
with  their  food.  As  a  result  of  this  treatment  the  animal  resists 


SUSCEPTIBILITY   AND   IMMUNITY.  263 

subcutaneous  injections  of  two  hundred  to  four  hundred  times  the 
fatal  dose  for  animals  not  having  this  artificial  immunity.  The  fatal 
dose  of  abrin  is  about  double  that  of  ricin.  When  injected  into  mice 
in  the  proportion  of  one  cubic  centimetre  to  twenty  grammes  of  body 
weight  a  solution  of  one  part  in  one  hundred  thousand  of  water 
proved  to  be  a  fatal  dose.  The  local  effects  are  also  less  pronounced 
when  solutions  of  abrin  are  used  ;  they  consist  principally  in  an  ex- 
tensive induration  of  the  tissues  around  the  point  of  injection  and  a 
subsequent  falling  off  of  the  hair  over  this  indurated  area.  When 
introduced  into  the  conjunctival  sac,  however,  abrin  produces  a 
local  inflammation  in  smaller  amounts  than  ricin,  a  solution  of  1 : 800 
being  sufficient  to  cause  a  decided  but  temporary  conjunctivitis. 
Solutions  of  1 :  50  or  1 : 100  of  either  of  these  toxalbumins,  introduced 
into  the  eye  of  a  mouse,  give  rise  to  a  panophthalmitis  which  com- 
monly results  in  destruction  of  the  eye.  But  in  mice  which  have 
been  rendered  immune  by  feeding  them  for  several  weeks  with  food 
containing  one  of  these  toxalbumins,  no  reaction  follows  the  intro- 
duction into  the  eye  of  the  strongest  possible  solution,  or  of  a  paste 
made  by  adding  abrin  to  a  little  ten-per-cent  salt  solution.  Ehrlich 
gives  the  following  explanation  of  the  remarkable  degree  of  im- 
munity established  in  his  experiments  by  the  method  mentioned: 

"  All  of  these  phenomena  depend,  as  may  be  easily  shown,  upon 
the  fact  that  the  blood  contains  a  body — antiabrin — which  completely 
neutralizes  the  action  of  the  abrin,  probably  by  destroying  this  body." 

In  a  more  recent  paper  Ehrlich  has  given  an  account  of  subse- 
quent experiments  which  show  that  the  young  of  mice  which  have 
an  acquired  immunity  for  these  vegetable  toxalbumins  may  acquire 
immunity  from  the  ingestion  of  the  mother's  milk  ;  and  also  that 
immunity  against  tetanus  may  be  acquired  in  a  very  brief  time  by 
young  mice  through  their  mother's  milk.  In  his  letanus  experi- 
ments Ehrlich  used  blood  serum  from  an  immune  horse  to  give  im- 
munity to  the  mother  mouse  when  her  young  were  .already  seven- 
teen days  old.  Of  this  blood  serum  two  cubic  centimetres  were 
injected  at  a  time  on  two  successive  days.  The  day  after  the  first 
injection  one  of  the  sucklings  received  a  tetanus  inoculation  by 
means  of  a  splinter  of  wood  to  which  spores  were  attached.  The 
animal  remained  in  good  health,  while  a  much  larger  control  mouse 
inoculated  in  the  same  way  died  of  tetanus  at  the  end  of  twenty-six 
hours.  Other  sucklings,  inoculated  at  the  end  of  forty-eight  and  of 
seventy-two  hours  after  the  mother  had  received  the  injection  of 
blood  serum,  likewise  remained  in  good  health,  while  other  control 
mice  died. 

The  possibility  of  conferring  immunity  by  means  of  the  milk  of 
an  immune  animal  is  further  shown  by  the  experiments  of  Brieger 


264  SUSCEPTIBILITY   AND    IMMUNITY. 

and  Ehrlich  (1892).  A  female  goat  was  immunized  against  tetanus 
by  the  daily  injection  of  "  thymus-tetanus  bouillon."  The  dose  was 
gradually  increased  from  0.2  cubic  centimetre  to  10  cubic  centimetres. 
At  the  end  of  thirty-seven  days  a  mouse,  which  received  0.1  cubic 
centimetre  of  the  milk  of  this  goat  in  the  cavity  of  the  abdomen, 
proved  to  be  immune  against  tetanus.  Further  experiments  gave  a 
similar  result,  even  when  the  milk  of  the  goat  was  not  injected  into 
the  peritoneal  cavity  of  the  mouse  until  several  hours  after  inocu- 
lation with  a  virulent  culture  of  the  tetanus  bacillus. 

When  the  casein  was  separated  the  milk  retained  its  full  im- 
munizing activity,  and  by  concentration  in  vacua  a  thick  milk 
was  obtained  which  had  a  very  high  immunization  value — 0.2  cubic 
centimetre  of  this  milk  protected  a  mouse  against  forty-eight  times 
the  lethal  dose  of  a  tetanus  culture. 

In  a  subsequent  communication  (1893)  Brieger  and  Ehrlich  de- 
scribe their  method  of  obtaining  the  antitoxin  of  tetanus  from  milk 
in  a  more  concentrated  form.  They  found  by  experiment  that  it  was 
precipitated  by  ammonium  sulphate  and  magnesium  sulphate.  From 
twenty-seven  to  thirty  per  cent  of  ammonium  sulphate  added  to  milk 
caused  a  precipitation  of  the  greater  part  of  the  antitoxin.  This  pre- 
cipitate was  dissolved  in  water,  dialyzed  in  running  water,  then 
filtered  and  evaporated  in  shallow  dishes  at  35°  C.  in  a  vacuum. 
One  litre  of  milk  from  an  immune  goat  gave  about  one  gramme  of  a 
transparent,  yellowish-white  precipitate,  which  contained  fourteen 
per  cent  of  ammonium  sulphate.  This  precipitate  had  from  four 
hundred  to  six  hundred  times  the  potency  of  the  milk  from  which 
it  was  obtained  in  neutralizing  the  tetanus  toxin. 

In  a  still  later  communication  (1893)  Brieger  and  Cohn  give  an 
improved  method  of  separating  the  antitoxin  from  the  precipitate 
thrown  down  with  ammonium  sulphate.  The  finely  pulverized  pre- 
cipitate is  shaken  up  with  pure  chloroform,  and  when  this  is  allowed 
to  stand  the  antitoxin  rises  to  the  surface  while  the  ammonium  salt 
sinks  to  the  bottom.  By  filling  the  vessel  to  the  margin  with  chloro- 
form, the  antitoxin  floating  on  the  surface  can  be  skimmed  off,  after 
which  it  quickly  dries.  By  this  method  the  considerable  loss  which 
occurred  in  the  dialyzer,  used  in  the  previously  described  method,  is 
avoided. 

A  most  interesting  question  presents  itself  in  connection  with  the 
discovery  of  the  antitoxins.  Does  the  animal  which  is  immune 
from  the  toxic  action  of  any  particular  toxalbumin  also  have  an  im- 
munity for  other  toxic  proteids  of  the  same  class?  The  experimental 
evidence  on  record  indicates  that  it  does  not.  In  Ehrlich 's  experi- 
ments with  ricin  and  abrin  he  ascertained  that  an  animal  which  had 
been  made  immune  against  one  of  these  subtances  was  quite  as  sus- 


SUSCEPTIBILITY    AND  "IMMUNITY.  265 

ceptible  to  the  toxic  action  of  the  other  as  if  it  did  not  possess  this 
immunity,  i.e.,  the  antitoxin  of  ricin  does  not  destroy  abrin,  and 
vice  versa.  As  an  illustration  of  the  fact,  he  states  that  in  one  ex- 
periment a  rabbit  was  made  immune  for  ricin  to  such  an  extent  that 
the  introduction  into  its  eye  of  this  substance  in  powder  produced  no 
inflammakny  reaction ;  but  the  subsequent  introduction  of  a  solution 
of  abrin,  of  1  to  10,000,  caused  a  violent  inflammation. 

Evidently  these  facts  are  of  the  same  order  as  those  relating  to 
immunity  from  infectious  diseases,  and,  taken  in  connection  with  the 
experimental  data  previously  referred  to,  give  strong  support  to  the 
view  that  the  morbid  phenomena  in  all  diseases  of  this  class  are  due 
to  the  specific  toxic  action  of  substances  resembling  the  toxalbumins 
already  discovered  ;  and  that  acquired  immunity  from  any  one  of 
these  diseases  results  from  the  formation  of  an  antitoxin  in  the  body 
of  the  immune  animal. 

Hankin  calls  these  substances  produced  in  the  bodies  of  immune 
animals  "defensive  proteids,"  and  proposes  to  classify  them  as  fol- 
lows :  First,  those  occurring  naturally  in  normal  animals,  which  he 
calls  sozins  ;  second,  those  occurring  in  animals  that  have  acquired 
an  artificial  immunity — these  he  calls  phylaxins.  Each  of  these 
classes  of  defensive  proteids  is  further  subdivided  into  those  which 
act  upon  the  pathogenic  microorganism  itself  and  those  which  act 
upon  its  toxic  products.  These  subclasses  are  distinguished  by  the 
prefixes  myco  and  toxo  attached  to  the  class  name. 

In  accordance  with  this  classification  a  mycosozin  is  a  defensive 
proteid,  found  in  the  body  of  a  normal  animal,  which  has  the  power 
of  destroying  bacteria. 

A  toxosozin  is  a  defensive  proteid,  found  in  the  body  of  a  normal 
animal,  which  has  the  power  of  destroying  the  toxic  products  of  bac- 
terial growth. 

A  inijcophylaxin  is  a  defensive  proteid  produced  in  the  body  of 
an  animal  which  has  an  acquired  immunity  for  a  given  infectious 
disease,  which  has  the  power  of  destroying  the  pathogenic  bacteria 
to  which  the  disease  is  due. 

A  toxophylaxin  is  a  defensive  proteid  produced  in  the  body  of 
an  animal  which  has  an  acquired  immunity  for  a  given  infectious 
disease,  which  has  the  power  of  destroying  the  toxic  products  of  the 
pathogenic  bacteria  to  which  the  disease  is  due. 

Buchner  had  previously  proposed  the  name  "  alexins  "  for  these 
defensive  proteids. 

The  importance  of  the  experimental  evidence  above  referred  to  in 
explaining  the  phenomena  of  natural  and  acquired  immunity  is  ap^ 
parent.  The  facts  stated  also  suggest  a  rational  explanation  of  re* 


266  SUSCEPTIBILITY   AND    IMMUNITY. 

covery  from  an  attack  of  an  acute  infectious  disease.  But  the  idea 
that  during  such  an  attack  an  antidote  to  the  disease  poison  is  de- 
veloped in  the  tissues  is  yet  so  novel,  and  the  experimental  evidence 
in  support  of  this  view  is  of  such  recent  date,  that  it  would  be  pre- 
mature to  accept  this  explanation  as  applying  to  immunity  in  gene- 
ral. It  seems  difficult  to  believe  that  a'n  individual  who  has  passed 
through  attacks  of  measles,  mumps,  whooping  cough,  scarlet  fever, 
small-pox,  etc.,  has  in  his  blood  or  tissues  a  store  of  the  antitoxin  of 
each  of  these  diseases,  formed  during  the  attack  and  retained  during 
the  remainder  of  his  life,  or  continuously  produced  so  long  as  the 
immunity  lasts.  Moreover,  in  those  diseases  to  which  the  experi- 
mental evidence  above  recorded  relates — diphtheria,  tetanus,  pneu- 
monia— as  they  occur  in  man,  no  lasting  immunity  has  been  shown 
to  result  from  a  single  attack,  and  in  this  regard  they  do  not  come 
into  the  same  class  with  the  eruptive  fevers  and  other  diseases  in 
which  a  single  attack  usually  protects  during  the  lifetime  of  the  in- 
dividual. 

In  those  instances  in  which  acquired  immunity  has  been  shown 
to  be  due  to  the  production  in  the  body  of  the  immune  animal  of  an 
antitoxin,  it  is  still  uncertain  whether  there  is  a  continuous  produc- 
tion of  the  protective  proteid,  or  whether  that  formed  during  the 
attack  remains  in  the  body  during  the  subsequent  immunity.  The 
latter  supposition  appears  at  first  thought  improbable  ;  but  when  we 
remember  that  the  protective  proteids  which  have  been  isolated  by 
Hankin  from  the  blood  and  spleen  of  rats,  and  by  Tizzoni  and  Cat- 
tani  from  the  blood  of  animals  made  immune  against  tetanus^  do 
not  dialyze,  it  does  not  seem  impossible  that  these  substances  might 
be  retained  indefimtely'within  the  blood-vessels.  On  the  other  hand, 
the  passage  of  the  tetanus  antitoxin  into  the  mother's  milk,  as 
shown  by  Ehrlich's  experiments  upon  mice,  indicates  a  continuous 
supply,  otherwise  the  immunity  of  the  mother  would  soon  be  lost. 

The  writer  has  obtained  (May,  1892)  experimental  evidence  that 
the  blood  of  vaccinated,  and  consequently  immune,  calves  contains 
something  which  neutralizes  the  specific  virulence  of  vaccine  virus, 
both  bovine  and  humanized.  Four  drops  of  blood  serum  from  a  calf 
which  had  been  vaccinated  two  weeks  previously,  mixed  with  one 
drop  of  liquid  lymph  recently  collected  in  a  capillary  tube,  after  con- 
tact for  one  hour  was  used  to  vaccinate  a  calf ;  the  same  animal  was 
also  vaccinated  with  lymph,  preserved  on  three  quills,  which  was 
mixed  with  four  drops  of  serum  from  the  immune  calf  and  left  for 
one  hour.  The  result  of  these  vaccinations  was  entirely  negative, 
while  vaccinations  upon  the  same  calf  made  with  virus  from  the 
same  source,  and  mixed  with  the  same  amount  of  blood  serum  from 
a  non-immune  calf,  gave  a  completely  successful  and  typical  result. 


SUSCEPTIBILITY    AND    IMMUNITY.  267 

The  experimental  evidence  detailed  shows  that  in  certain  dis- 
eases acquired  immunity  depends  upon  the  formation  of  anti- 
toxins in  the  bodies  of  immune  animals.  As  secondary  fac- 
tors it  is  probable  that  tolerance  to  the  toxic  products  of  pathogenic 
bacteria  and  phagocytosis  have  considerable  importance,  but  it  is 
evident  that  the  principal  role  cannot  be  assigned  to  these  agencies. 

As  a  rule  the  antitoxins  have  no  bactericidal  action;  but  it  has 
been  shown  by  the  experiments  of  Gamaleia,  Pfeiffer,  and  others, 
that  in  animals  which  have  an  acquired  immunity  against  the  spiril- 
lum of  Asiatic  cholera  and  against  spirillum  Metschnikovi,  there  is  a 
decided  increase  in  the  bactericidal  power  of  the  blood  serum,  and 
that  immunity  probably  depends  upon  this  fact.  9 

The  researches  of  Metschnikoff  upon  hog  cholera,  of  Issaef  upon 
pneumonia,  and  of  Sanarelli  upon  typhoid  fever  indicate  that  the 
immunity  conferred  upon  susceptible  animals  by  protective  inocula- 
tions is  not  due  to  an  antitoxin  but  to  a  substance  present  in  the 
blood  of  immune  individuals  which  acts  directly  upon  the  pathogenic 
microorganism,  as  is  the  case  in  cholera-immune  animals.  The  ani- 
mals immunized  are  said  to  be  quite  as  sensitive  to  the  action  of  the 
bacterial  poisons  as  are  those  which  have  not  received  protective 
inoculations.  "Their  serum  does  not  protect  against  the  toxin,  but 
against  the  microbe"  (Roux) . 

According  to  Buchner  (1894)  the  antitoxins  are  to  be  regarded  not 
as  reactive  products  developed  in  the  body  of  the  immune  animal, 
but  as  modified,  changed,  and  "  entgiftete  "  products  of  the  specific 
bacterial  cells.  He  insists  that  they  do  not  neutralize  the  toxins  by 
direct  contact,  but  only  through  the  medium  of  the  living  organism. 
This  explanation  scarcely  appears  tenable  in  view  of  the  experimental 
evidence,  and  the  fact  that  the  antitoxin  of  tetanus  escapes  in  con- 
siderable quantity  with  the  milk  of  an  immune  goat  without,  ap- 
parently, diminishing  the  immunity  of  the  animal.  In  the  immunity 
against  the  toxic  action  of  the  vegetable  toxalbumins — ricin  and 
abrin — as  shown  by  Ehrlich's  experiments,  there  are  no  "  products  of 
bacterial  cells  "  introduced  with  the  pure  toxalbumin  from  the  castor 
bean  or  the  jequirity  bean ;  and  we  have  sufficiently  numerous  ex- 
periments to  show  that  immunity,  with  the  presence  of  antitoxins  in 
the  blood,  may  be  induced  by  precipitated  and  purified  toxalbumins 
from  filtered  cultures.  Several  of  the  experimenters,  also,  have  re- 
ported that  the  toxins  from  bacterial  cultures  are  neutralized  in  vitro 
by  blood-serum  from  an  immune  animal,  or  by  the  precipitated  anti- 
toxin from  such  serum  after  contact  for  a  certain  number  of  hours. 
If  they  are  correct  in  the  statement  that  a  certain  time  is  required 
after  the  antitoxin  has  been  brought  in  contact  with  the  toxin,  in 
order  that  the  latter  may  be  neutralized,  as  shown  by  injection  of  the 


268  SUSCEPTIBILITY   AND   IMMUNITY. 


mixture  into  a  susceptible  animal,  then  we  must  admit  that  this 
neutralizing  effect  occurs  outside  of  the  body  of  the  animal,  as  has 
been  generally  assumed. 

The  experiments  of  Vaillard  are  also  opposed  to*Buchner's  view. 
He  reports  that  in  a  rabbit  immunized  against  tetanus,  "  a  vohfme  of 
blood  equal  to  the  total,  amount  which  circulates  in  its  body  may  be 
withdrawn  without  diminishing,  in  an  -appreciable  manner,  the  anti- 
toxic power  of  its  serum.  Therefore  the  antitoxin  must  be  repro- 
duced as  fast  as  it  is  withdrawn."  The  author  from  whom  we  have 
just  quoted  (Roux)  also  reports  the  resujts  of  experiments  which 
show  that  the  antitoxic  value  of  the  serum  of  a  rabbit  immunized 
against  tetanus  does  not  bear  a  direct  relation  to  the  quantity  of  the 
tetanus  toxin-  introduced,  but  depends  also  upon  the  method  adopted. 
W^ien  a  few  large  doses  are  given  the  result  is  far  lesg  favorable  than 
that  obtained  by  giving  the  same  amount  in  repeated  small  doses. 
The  serum  of  an  animal  immunized  by  thirty-three  small  doses  was 
found  to  neutralize,  in  vitro,  150  parts*of  toxin,  while  that  of  an 
animal  which  received  the  same  amount  in  nine*  doses  only  neutral- 
ized 25  parts  of  the  same  toxin.  On  the  other  hand  we  have  experi- 
ments which  indicate  that  the  supposed  neutralization  of  a  toxin  by 
an  antitoxin  in  vitro  is  not  really  a  chemical  neutralization.  Thus 
Buchner  found  in  his  experiments  with  the  tetanus  toxin  and  Anti- 
toxin, in  a  dry  powder,  that  when  mixed  in  a  certain  proportion  and 
injected  into  white  mice  no  tetanic  symptoms  were  induced.  But  the 
same  mixture  gave  rise  to  distinct  tetanic  symptoms  in  guinea-pigs, 
showing'that  the  inference  that  the  toxin  had  been  neutralized  in 
vitro,  based  upon  the  experiment  on  mice,  would  have  been  a  mis- 
take. And  certain  observations  made  by  Roux  and  Vaillard  seem 
to  give  support  to  the  view  that  neutralization  does  not  occur  in 
vitro,  but  that  the  result  depends  upon  some  physiological  reaction 
induced  by  the  antitoxin  within  the  body  of  the  living  animal.  These 
bacteriologists  found  that  when  the  antitoxin  was  apparently  in  ex- 
cess, tetanic  symptoms  could*  be  induced  in  susceptible  animals  if 
they  had  been  in  any  way  exhausted  prior  to  the  injection  of  the 
mixture  of  toxin  -and  antitoxin;  and  that  the  same  result  followed 
when  their  resisting  power  had  been  reduced  by  injecting  into  them 
at  the  same  time  filtered  cultures  of  other  bacteria. 

In  this  connection  the  results  reported  by  Calmette,  Phisalix, 
and  Bertrand  are  of  interest.  These  investigators  found  that  when 
the  antitoxin  of  snake-poison  was  mixed  with  this  venom  in  a  pro- 
portion which  neutralized  its  toxic  properties,  as  shown  by  experi- 
mental inoculations,  and  the  mixture  then  heated  to  70°  C.,  by  which 


SUSCEPTIBILITY   AND   IMMUNITY.  269 

temperature  the  antitoxin  is  destroyed,  subsequent  inoculations 
showed  that  the  toxin  was  still  active. 

The  experiments  of  Stern  (1894)  show  that  the  typhoid  bacil- 
lus not  only  grows  in  blood-serum  from  a  typhoid  convalescent,  which 
has  been  proved  to  neutralize  its  pathogenic  effects  when  injected 
into  a  susceptible  animal,  but  also  that  its  toxic  products  are  de- 
veloped in  this  culture  medium.  From  this  Stern  concludes  that  the 
serum  must  in  some  way  act  upon  the  infected  animal,  causing 
changes  which  enable  it  to  resist  infection,  rather  than  upon  the 
bacillus  or  upon  its  toxic  products  directly.  It  has  also  been  shown 
by  Behring  (1890)  for  the  diphtheria  bacillus,  by  Vaillard  for  the 
tetanus  bacillus  (1892),  and  by  Issaeff  (1893)  for  the  micrococcus  of 
pneumonia,  that  these  several  pathogenic  microorganisms  may  be 
cultivated  in  the  blood-serum  of  animals  immunized  for  the  diseases 
which  they  produce. 

In  a  paper  published  in  1897,  Ehrlich  advanced  his  "side-chain" 
(seitenkette)  theory.  He  considers  the  individual  cells  of  the  body  to 
be  analogous,  in  a  certain  sense,  to  complex  organic  substances,  and 
that  they  consist  essentially  of  a  central  nucleus  to  which  secondary 
atom-groups  having  distinct  physiological  functions  are  attached  by 
"  side  chains  "  —such  as  chemists  represent  in  their  attempts  to  illus- 
trate the  reactions  which  occur  in  the  building  up  or  pulling  down  of 
complex  organic  compounds.  The  cell-equilibrium  is  supposed  to  be 
disturbed  by  injury  to  any  of  its  physiological  atom-groups — as  by  a 
toxin — and  this  disturbance  results  in  an  effort  at  compensatory  repair 
during  which  plastic  material  in  excess  of  the  amount  required  is 
generated  and  finds  its  way  into  the  blood.  This  Ehrlich  regards  as 
the  antitoxin,  which  is  capable  of  neutralizing  the  particular  toxin 
to  which  it  owes  its  origin,  if  this  is  subsequently  introduced  into  the 
blood.  In  this  theory  a  specific  combining  relation  is  assumed  to 
exist  between  various  toxic  substances  and  the  secondary  atom- 
groups  of  certain  cellular  elements  of  the  body.  The  atom-groups 
which,  in  accordance  with  this  theory,  combine  with  the  toxin  of  any 
particular  disease  germ,  Ehrlich  calls  the  "toxophoric  side  chain." 
Immunity,  according  to  Ehrlich,  is  either  "active"  or  "passive." 
Passive  immunity  results  from  the  introduction  of  the  immunizing 
substance  from  an  immunized  animal  into  the  circulation  of  a  non- 
immune  animal,  e.g.,  the  use  of  diphtheria  antitoxin  as  a  prophy- 
lactic. This  passive  immunity  is  more  transient  than  the  active 
immunity  which  results  from  an  attack  of  an  infectious  disease,  from 
inoculations  with  living  vaccines,  or  from  repeated  injections  of  in- 
creasing doses  of  the  toxins  of  pathogenic  bacteria.  Ehrlich's  ex- 


270  SUSCEPTIBILITY   AND   IMMUNITY. 

planation  of  immunity,  however  probable  it  may  appear,  can  hardly 
be  said  to  rest  upon  a  substantial  experimental  foundation,  and  we 
must  admit  that  the  exact  source  and  method  of  production  of  the 
antitoxins  in  the  animal  body,  and  their  mode  of  action,  are  still 
undetermined ;  and,  for  the  present,  we  must  be  satisfied  with  the 
knowledge  that  in  some  way  these  so-called  antitoxins,  which  have 
been  proved,  to  be  present  in  the  blood-serum  of  immune  animals, 
protect  these  animals  from  infection  by  pathogenic  bacteria.  And 
that  when  transferred  to  susceptible  animals  they  confer  upon  them 
a  temporary  immunity ;  or  if  introduced  after  infection,  may  neutral- 
ize the  pathogenic  action  of  the  toxins  produced  by  specific  "disease 
germs." 

Finally,  there  is  experimental  evidence  to  show  that  immunity 
from  the  pathogenic  action  of  certain  bacteria  may  be  produced  by 
previous  injections  of  cultures  of  other  bacteria  (sterilized  or  other- 
wise), and  even  by  the  injection  of  the  blood-serum  of  normal  indi- 
viduals or  of  other  substances. 

Pasteur'  in  1880,  communicated  to  the  French  Academy  of  Sciences 
the  results  of  experiments  which  led  him  to  the  conclusion  that  fowls 
which  had  an  acquired  immunity  against  chicken  cholera  also  had 
an  immunity  against  anthrax.  Roux  has  reported  that  the  blood- 
serum  of  a  horse  which  has  been  immunized  against  tetanus  neutral- 
izes the  toxic  power  of  cobra  poison.  But  the  contrary  effect  is  not 
produced — i.e.,  the  blood-serum  of  an  animal  immunized  against  the 
cobra  poison  does  not  neutralize  the  tetanus  toxalbumin.  The  state- 
ment is  also  made  that  the  blood-serum  of  a  rabbit  which  has  been 
made  immune  against  hydrophobia  will  protect  a  susceptible  animal 
against  the  cobra  venom  in  doses. four  or  five  times  as  large  as  the 
usually  lethal  dose.  Also  that  rabbits  which  have  been  immunized 
against  snake-poison  are  less  susceptible  to  the  toxic  effects  of  abrin, 
and  the  reverse — i.e.,  antiabrin  neutralizes,  to  some  extent  at  least, 
the  toxic  action  of  snake-poison. 

The  writer,  in  his  "  Report  on  the  Etiology  and  Prevention  of 
Yellow  Fever"  (1890),-  gives,  on  pp.  196  and  197,  experimental  evi- 
dence which  shows  that  the  injection  into  the  peritoneal  cavity  of 
rabbits  of  cultures  of  Bacillus  pyocyaneus  or  of  Bacillus  gracilis  pro- 
tected the  animals  from  the  fatal  results  of  subsequent  injections  of 
my  bacillus  X,  which  was  extremely  fatal  to  rabbits  when  injected 
into  the  cavity  of  the  abdomen  in  doses  of  1  or  2  c.c.  In  referring 
to  these  experiments  I  say :  "  The  evidence  favors  the  view  that  death 
results  from  peritonitis  (and  toxemia?)  induced  by  intr'a-peritoneal 
injections,  and  that  a  tolerance  on  the  part  of  the  peritoneum  may 


PLATE   IV. 

FIGS.  1,  2,  and  3. — Leucocytes  from  the  spleen  of  an  inoculated 
monkey,  containing  Spirillum  Obermeieri.  (Soudakewitch.) 

FIGS.  4  and  5. —Leucocytes  ("  niacrophages  ")  from  a  preparation  of 
muscle  from  a  pigeon  which  succumbed  to  an  anthrax  inoculation.  In 
Fig.  4  the  bacilli  are  deeply  stained;  in  Fig.  5  they  are  pale.  (Metsch- 
nikoff.) 

FIG.  6. — Leucocyte  from  a  frog  seventy-two  hours  after  the  injection 
of  anthrax  spores.  (Trapeznikoff . ) 

FIGS.  7  and  8. — Leucocytes  from  a  chicken  four  hours  after  the  in- 
jection of  anthrax  spores.  (Trapeznikoff.) 


ERNBERG'S  BACTERIOLOGY. 


Plate  F 


Fig.l. 


Fig.  2. 


Fig  3. 


Fig.  5. 


Fig.  7. 


PHAGOCYTES. 


SUSCEPTIBILITY   AND    IMMUNITY.  271 

be  established  by  the  injection  of  certain  other  bacilli,  or  of  ster- 
ilized cultures  of  bacillus  X." 

This  corresponds  with  facts  subsequently  developed  by  Issaeff 
(1894)  in  his*  experiments  with  reference  to  immunity  in  guinea- 
pigs  against  cholera  cultures  injected  into  the  cavity  of  the  abdomen. 
He  found  that  a  certain  degree  of  immunity  was  established  by  the 
previous  injection  of  blood-serum  from  normal  individuals',  and  'also 
of  various  acids,  alkalies,  and  neutral  liquids.  The  immunity  pro- 
duced in  this  way  was, .  however,  feeble  and  temporary,  and  could 
not  properly  be  considered  as  identical  with  that  produced  by  inocula- 
tions with  attenuated  cultures  which  give  rise  to  a  mild  attack  of  a 
specific  disease. 

Cesaris-Demel  and  Orlandi  have  reported  (1894)  their  success  in 
immunizing  animals  against  infection  by  the  typhoid  bacillus  by 
means  of  sterilized  cultures  of  Bacillus  coli  communis,  and  the 
reverse. 

While  this  chapter  relates  especially  to  acquired  immunity  from 
infectious  diseases,  and  this  immunity  has  been  shown  to  depend,  in 
a  number  of  these  diseases  at  least,  upon  the  development  of  anti- 
toxins in  the  body  of  the  immune  animal,  it  may  be  worth  while  to 
refer  briefly,  before  closing,  to  some  examples  of  acquired  immunity 
of  a  different  order,  We  refer  to  the  tolerance  to  extremes  of  heat 
and  cold  which  may  be  established  by  habitual  exposure,  and,  more 
especially,  to  the  tolerance  to  narcotics  and  irritant  poisons,  which  is 
very  remarkable  and  has  never  been  explained  in  a  satisfactory 
manner.  Samuel  (1*92)  has  presented  experimental  evidence  which 
shows  that  the  local  inflammation  which  results  from  the  application 
of  croton-oil  to  the  ear  of  a  rabbit  does  not  occur  when  a  second  ap- 
plication is  made  to  the  same  ear  after  recovery  from  the  effects  of 
the  first.  That  a  tolerance  may  be  acquired  to  comparatively  large 
doses  of  arsenic  is  well  known,  and  the  tolerance  which  the  victims 
of  drug  habits  acquire  to  enormous  doses  of  narcotics  is  a  matter  of 
daily  observation.  In  the  writer's  paper  on  acquired  immunity,  pub- 
lished in  1881,  an  attempt  was  made  to  account  for  acquired  im- 
munity in  infectious  diseases  as  analogous  to  the  immunity  to  drugs » 
just  referred  to ;  but  the  experimental  evidence  presented  in  the  pres- 
ent chapter  shows  that  the  analogy  has  no  scientific  foundation  in 
the  absence  of  any  evidence  that  there  is  an  antitoxin  of  morphia,  of 
cocaine,  of  narcotin,  etc.,  in  the  blood  of  the  habitues  of.  these  drugs. 


IV. 
PKOTECTIVE  INOCULATIONS. 

ANTHRAX. 

THE  discovery  of  the  anthrax  bacillus  by  Davaine  (1863),  and  the 
demonstration  of  its  etiological  relation  to  the  disease  with  which  it  is 
associated,  by  the  researches  of  Pasteur,  Toussaint,  Koch  (1878-1881) 
and  other  pioneers  in  this  field  of  investigation,  constitute  the  foun- 
dation of  our  present  knowledge  of  bacteriology  and  of  the  practical 
results  attained  in  protective  inoculations  and  serum-therapy.  And  a 
review  of  the  literature  relating  to  the  anthrax  bacillus  would  show, 
in  a  most  interesting  manner,  the  successive  steps  by  which  we  have 
arrived  at  the  important  results  which  have  gone  so  far  toward  estab- 
lishing medicine  upon  a  scientific  basis.  In  the  present  volume, 
however,  we  must  confine  our  attention  to  those  investigations  which 
relate  directly  to  the  subject  in  hand. 

Toussaint,  a  pioneer  in  researches  relating  to  protective  inocula- 
tions, has  a  short  paper  in  the  Comptes-Rendm  of  the  French  Academy 
'of  Sciences  of  July  12th,  1880,  entitled  "Immunity  from  Anthrax 
("charbon")  Acquired  as  a  Result  of  Protective  Inoculations." 

In  this  paper  he  announces  his  discovery  of  the  important  fact  that 
the  anthrax  bacillus  does  not  form  spores  in  the  tissues  or  liquids  of 
the  body  of  an  infected  animal,  but  multiplies  alone  by  binary  divi- 
sion— " sa  multiplication  sefait  toujaurspar  une  division  du  mycelium." 

In  the  same  communication  he  reports  his  success  in  conferring 
immunity  upon  five  sheep  by  means  of  protective  inoculations,  and 
also  upon  four  young  dogs.  We  must  therefore  accord  him  the  prior- 
ity in  the  publication  of  experimental  data  demonstrating  the  practi- 
cability of  accomplishing  this  result. 

Toussaint  does  not  give  his  method  in  the  communication  above 
referred  to,  but  the  following  quotation  from  a  communication  made 
to  the  Academy  of  Sciences  on  March  19th,  1881,  by  Pasteur,  shows 
the  method,  and  at  the  same  time  demonstrates  the  fact  that  Tous- 
saint was  the  first  to  produce  immunity  by  the  use  of  sterilized  cul- 
tures. Pasteur  says : 

"  By  inoculating  sheep  either  with  defibrinated  blood  from  an  animal 
dead  of  anthrax,  after  nitration  through  several  thicknesses  of-  paper,  or 


PROTECTIVE   INOCULATIONS.  273 

with  the  same  blood  defibrinated  and  subjected  to  55  C.  for  ten  minutes, 
according-  to  Toussaiiit,  these  sheep  subsequently  resist  inoculations  with 
anthrax  blood.  .  .  .  The  bacillus,  according-  to  Toussaint,  deposits  in  the 
blood  of  animals  in  which  it  multiplies  a  substance  which  may  become  its 
own  vaccine.  By  filtration  while  cold  in  one  case,  by  a  temperature  of  55 c  C. 
in  the  other,  the  bacillus  is  said  to  be  removed  or  killed  ;  so  that  the  inocula- 
tion of  filtered  or  heated  blood  introduces  into  the  animal  inoculated  vac- 
cinal  matter  deprived  of  bacteria." 

After  thus  stating  Toussaiut's  method  and  explanation  Pasteur 
proceeds  to  raise  objections  against  this  method,  the  principal  of 
which  are  that  the  anthrax  bacillus  is  not  killed  by  exposure  to  a  tem- 
perature of  55°  C.  for  ten  minutes,  and  that  inoculation  with  a  virus 
prepared  in  this  way  would  result  in  a  considerable  mortality  among 
the  animals  inoculated,  although  those  surviving  the  inoculation  would 
be  protected. 

In  a  communication  made  to  the  French  Academy  of  Sciences, 
September  27th,  1880,  Pasteur  gave  an  account  of  an  experiment  made 
July  14th,  1879,  upon  two  cows,  which  in  connection  with  a  subsequent 
experiment,  made  August  6th,  1880,  upon  four  cows,  led  him  to  the 
conclusion  that  a  single  attack  of  anthrax  protects  from  subsequent 
attacks.  He  says  in  the  paper  referred  to : 

"On  the  15th  of  September,  1880,  two  cows,  A  and  C,  which  had  been 
very  ill  as  a  result  of  the  first  inoculation,  made  August  6th,  were  reinoculated 
on  the  left  side,  that  is  to  say,  on  the  side  opposite  the  first  inoculation.  We 
used  five  drops  of  culture  of  the  bacillus  of  anthrax  ('  bacte-ridies  du  char- 
bon"1).  The  following-  days  there  was  no  perceptible  cedema  and  no  elevation 
of  temperature  in  either  cow.  The  question  is  then  resolved  :  a  single  attack 
protects  ('le  charbon  ne  r&cidive  pas'1)." 

The  next  important  steps  in  the  line  of  experimental  research 
leading  to  protective  inoculations  in  the  disease  under  consideration 
were  reported  by  Pasteur  in  his  communication  to  the  French  Acad- 
emy made  at  the  seance  of  February  28th,  1881  (with  the  collaboration 
of  Chamberland  and  Roux),  entitled  "De  1' attenuation  des  virus  et 
de  leur  retour  a  la  virulence. "  In  this  connection  Pasteur  announces 
his  discovery  of  the  fact  that  when  cultivated  at  a  temperature  of  42° 
to  43°  C.,  the  anthrax  bacillus  no  longer  forms  spores  and  rapidly 
loses  its  virulence.  He  says :  ^ 

'  'As  regards  its  virulence,  the  extraordinary  fact  has  been  ascertained  that 
the  bacillus  is  no  longer  virulent  after  it  has  been  kept  for  eight  days  at  a 
temperature  of  42  to  43"  C.  ;  at  least  its  cultures  are  inoffensive  for  the 
guinea-pig,  the  rabbit,  and  the  sheep,  three  species  of  animals  which  are 
very  susceptible  to  anthrax.  We  are  able,  then,  not  only  to  attenuate  viru- 
lence, but  to  effect  its  complete  extinction,  by  a  simple  method  of  cultivation. 

"Before  the  extinction  of  its  virulence  the  microbe  of  charbon  passes 
through  the  intermediate  degrees  of  attenuation,  and,  on  the  other  hand,  as 
happens  also  with  the  microbe  of  fowl  cholera,  each  of  these  degrees  of  vir- 
18 


274  PROTECTIVE   INOCULATIONS. 

ulence  may  be  reproduced  by  cultivation.  Finally,  as  shown  in  one  of  our 
recent  communications,  since  one  attack  of  anthrax  protects,  each  one  of  our 
attenuated  microbes  of  charbon  constitutes  a  vaccine  for  the  microbe  of 
superior  virulence  ;  that  is  to  say,  a  virus  suitable  to  produce  a  more  benign 
malady.  What,  then,  is  more  easy  than  to  find  among  these  a  virus  suitable 
to  give  anthrax  to  sheep,  cows,  or  horses,  without  causing  them  to  perish, 
and  capable  of  preserving  them  from  a  subsequent  fatal  attack  ?  We  have 
already  practised  this  operation  upon  sheep  with  great  success." 

At  the  end  of  this  important  communication  Pasteur  says : 

"I  concluded  my  communication  of  October  26th  by  remarking  that  the 
attenuation  of  virus  by  the  influence  of  the  air  is  probably  one  of  the  factors 
in  the  extinction  of  great  epidemics.  The  facts  just  recorded,  in  their  turn, 
may  serve  to  explain  the  so-called  spontaneous  appearance  of  these  scourges. 
An  epidemic  which  has  been  terminated  by  the  attenuation  of  its  virus  may 
be  relighted  by  the  reinforcement  of  this  virus  under  certain  influences.  The 
accounts  which  I  have  read  of  the  spontaneous  appearance  of  the  plague  ap- 
pear to  me  to  offer  examples  of  this.  The  plague  is  a  virulent  malady  which 
prevails  in  certain  countries.  In  all  of  these  countries  its  attenuated  virus 
probably  exists,  ready  to  take  its  active  form  when  the  necessary  conditions 
as  to  climate,  famine,  and  distress  again  prevail.  There  are  other  virulent 
maladies  which  appear  spontaneously  in  all  countries,  such  as  camp  typhus. 
Without  doubt  the  germs  of  the  microbes  which  cause  these  diseases  are 
everywhere  distributed.  Man  carries  them  about  him,  or  in  his  intestine, 
without  great  damage,  but  ready,  nevertheless,  to  become  dangerous  when, 
as  a  result  of  certain  conditions  or  of  successive  development  upon  the  sur- 
face of  wounds,  in  bodies  enfeebled  or  otherwise,  their  virulence  is  progres- 
sively reinforced.  And  from  this  point  of  view  virulence  appears  to  us 
under  a  new  light  which  is  somewhat  disquieting  for  humanity,  unless  na- 
ture, in  the  evolution  which  has  occurred  during  the  past  centuries,  has  al- 
ready encountered  all  possible  occasions  for  the  production  of  virulent  or 
contagious  diseases,  an  assumption  which  seems  very  improbable. 

"  What  is  an  inoffensive  microscopic  organism  for  man  or  for  a  given 
animal  ?  It  is  an  organism  which  cannot  develop  in  our  body  or  in  that  of 
the  animal ;  but  nothing  proves  that  if  this  microscopic  organism  should 
penetrate  into  some  other  of  the  thousands  of  species  of  the  creation,  it  could 
not  invade  it  and  cause  it  to  become  sick.  Its  virulence,  then,  reinforced  by 
passing  through  a  series  of  individuals  of  this  species,  might  become  such 
that  it  could  invade  man  or  one  of  the  domestic  animals.  By  this  means  new 
contagions  may  be  created.  I  am  disposed  to  believe  that  it  is  in  this  way 
that,  in  the  course  of  ages,  have  appeared  small-pox,  syphilis,  the  plague, 
yellow  fever,  etc." 

This  broad  induction  has  received  considerable  support  from  more 
recent  researches,  which  show  that  the  typhoid  bacillus,  the  cholera 
spirillum,  and  other  important  pathogenic  bacteria  become  attenuated 
when  they  lead  a  saprophytic  existence  for  some  time,  and  regain  their 
virulence  when  they  are  propagated  within  the  bodies  of  susceptible 
animals. 

In  a  later  communication  (March  21st,  1881)  Pasteur  says  that  he 
has  found  by  experiment  that  when  attenuated  varieties  of  the  anthrax 
bacillus  form  spores,  these  again  reproduce  the  same  pathogenic  va- 
riety, so  that  cultures  of  each  degree  of  attenuation  can  be  maintained 
indefinitely. 


PROTECTIVE   INOCULATIONS.  275 

On  June  13th.  1881.  Pasteur  communicated  the  results  of  his  famous 
experiment  at  Pouilly-le-Fort,  near  Melun.  He  says : 

4 'On  the  5th  of  May.  1881,  we  inoculated,  by  means  of  a  Pravez  syringe, 
twenty-four  sheep,  one  goat,  and  six  cows,  each  animal  with  five  drops  of 
an  attenuated  culture  of  the  anthrax  bacillus.  On  the  17th  of  May  we  rein- 
oculated  these  animals  with  a  second  virus,  also  attenuated,  but  more  virulent 
than  the  first. 

"On  the  31st  of  May  we  proceeded  to  make  a  very  virulent  inoculation  in 
order  to  test  the  efficacy  of  the  preventive  inoculations  made  011  the  5th  and 
17th  of  May.  For  this  experiment  we  inoculated  the  vaccinated  animals, 
and  also  twenty-four  sheep,  one  goat,  and  four  cows  which  had  not  received 
any  previous  treatment. 

"The  very  virulent  virus  used  on  the  31st  of  May  was  obtained  from 
spores  preserved  in  my  laboratory  since  the  21st  of  March,  1877. 

"In  order  to  make  the  experiments  more  comparable  we  inoculated  alter- 
nately a  vaccinated  and  a  non-vaccinated  animal.  When  the  operation  was 
finished  all  of  those  present  were  invited  to  reassemble  011  June  2d,  i.e., 
forty-eight  hours  after  the  virulent  inoculation  \vas  made. 

"  Upon  the  arrival  of  the  visitors  on  June  2d,  all  were  astonished  at  the 
result.  The  twenty-four  sheep,  the  goat,  and  the  six  cows  which  had  received 
the  attenuated  virus,  all  presented  the  appearance  of  health.  On  the  con- 
trary, twenty  of  the  sheep,  and  the  goat,  which  had  not  been  vaccinated,  were 
already  dead  of  anthrax;  two  more  of  the  11011- vaccinated  sheep  died  before 
the  eyes  of  the  spectators,  and  the  last  of  the  series  expired  before  the  end  of 
the  day.  The  non -vaccinated  cows  were  not  dead.  We  had  previously 
proved  that  cows  are  less  subject  than  sheep  to  die  of  anthrax.  But  all  had 
an  extensive  oedema  at  the  point  of  inoculation,  behind  the  shoulder.  Cer- 
tain of  these  oedematous  swellings  increased  during  the  following  days  to  such 
dimensions  that  they  contained  several  litres  of  liquid,  deforming  the  animal. 
One  of  them  even  nearly  touched  the  earth.  The  temperature  of  these  cows 
was  elevated  3  C.  The  vaccinated  cows  did  not  experience  any  elevation  of 
temperature,  or  tumefaction,  or  the  slightest  loss  of  appetite.  The  success, 
therefore,  was  as  complete  for  the  cows  as  for  the  sheep."" 

The  facts  that  infection  depends  to  some  extent  upon  the  number 
of  bacilli  introduced,  and  that  animals  which  have  a  certain  degree  of 
immunity,  like  the  Algerian  race  of  sheep,  may  succumb  when  they 
are  inoculated  with  a  certain  quantity  of  virus,  although  they  resist 
a  smaller  amount,  were  announced  by  Chauveau  in  his  communication 
to  the  French  Academy  at  the  seance  of  June  28th,  1880.  He  says : 

"The  facts  which  I  have  just  presented  show  that  the  anthrax  bacillus 
behaves  in  the  organism  of  Algerian  sheep,  not  as  if  it  were  deprived  of  the 
principles  necessary  for  its  development,  but  rather  as  if  it  were  in  a  medium 
rendered  unsuitable  for  its  growth  by  the  presence  of  substances  injurious  to 
it.  In  a  very  small  number  the  bacilli  are  arrested  in  their  development  by 
the  inhibitory  influence  of  these  substances.  When  they  are  very  numerous, 
on  the  contrary,  they  surmount  more  easily  this  obstacle  to  their  prolifera- 
tion." 

This  quotation  shows  that  Chauveau  had  at  this  early  date  arrived 
at  an  explanation  of  immunity  very  nearly  in  accord  with  that  which 
is  now  generally  accepted. 

The  fact  that  infection  is  influenced  by  the  quantity  of  the  infec- 


276  PROTECTIVE   INOCULATIONS. 

tious  material  introduced  had  previously  been  insisted  upon  by  Da- 
vaine  in  his  paper  entitled  "  Eeclierclies  sur  quelques  lines  des  conditions 
qui  favorisent  ou  qui  empechent  le  development  de  la  septicemie,"  pub- 
lished in  the  Bulletin  of  the  Academy  of  Medicine,  seance  of  February 
18th,  1879. 

Davaine  says : 

"A  third  condition  relates  to  the  quantity  of  bacteria  introduced  into  the 
tissues.  This  question  of  quantity  has  been  made  manifest  in  our  experi- 
ments. Not  only  does  it  differ  in  different  species  of  animals,  the  rabbit  and 
the  dog,  for  example,  but  it  varies  in  different  animals  of  the  same  species." 

In  his  communication  to  the  Academy  of  Sciences,  made  on  April 
4th,  1881,  Chauveau  gives  the  results  of  his  experiments  in  producing 
immunity  by  inoculations  with  very  small  quantities  of  virus.  After 
some  preliminary  experiments  with  a  larger  number,  five  sheep  were 
inoculated  with  diluted  anthrax  blood  estimated  to  contain  two  hun- 
dred and  fifty  bacilli  for  each.  All  of  the  animals  survived  the  inocu- 
lation after  having  manifested  some  slight  febrile  reaction.  Six  weeks 
later  all  were  reinoculated  with  a  dose  which  should  have  been  fatal 
to  an  unprotected  animal.  One  of  the  animals  died  of  anthrax,  the 
other  four  resisted  perfectly. 

On  June  26th,  1882,  Chauveau  reported  to  the  Academy  of  Sciences 
the  results  of  his  experiments  relating  to  the  protection  of  animals 
from  anthrax  infection  by  the  method  of  Toussaint.  By  carefully 
conducted  experiments  Chauveau  found  that  nine  or  ten  minutes'  ex- 
posure to  a  temperature  of  54°  C.  killed  all  of  the  bacilli  in  anthrax 
blood,  and  the  same  result  was  obtained  by  sixteen  minutes'  exposure 
to  52°  C.,  while  at  50°  C.  the  time  required  is  twenty  minutes.  An 
attenuated  virus  suitable  for  protective  inoculations  is  obtained  by 
exposure  for  a  somewhat  shorter  time,  and  as  a  result  of  his  experi- 
ments Chauveau  was  led  to  the  conclusion  that  for  a  first  inoculation 
anthrax  blood  heated  to  50°  C.  for  fifteen  minutes  afforded  a  good  at- 
tenuated virus.  This  was  to  be  followed  after  an  interval  of  ten  to 
fifteen  days  by  a  second  inoculation  with  a  stronger  virus,  obtained 
by  exposing  anthrax  blood  to  the  same  temperature  (50°  C.)  for  nine 
or  ten  minutes.  These  inoculations  sufficed  to  protect  the  animals 
when  they  were  subsequently  inoculated  with  virus  of  full  strength — 
blood  from  an  animal  which  had  recently  succumbed  to  the  disease. 
Chauveau  says  with  reference  to  this  method : 

"  In  one  hour,  with  a  single  guinea-pig  [dead  of  anthrax],  it  is  easy  to 
prepare  the  quantity  of  vaccine  required  to  inoculate  more  than  five  hundred 
sheep.  The  inoculation  is  made  with  the  point  of  a  lancet,  charged,  by  the 
method  in  use  in  my  laboratory,  with  a  very  small  quantity  of  virus.  Two 


PROTECTIVE   INOCULATIONS.  277 

or  three  large  punctures  under  the  skin,  upon  the  internal  surface  of  the  ear, 
suffice  for  a  successful  inoculation. 

' '  The  vaccine  prepared  in  this  way  should  be  used  at  once,  or  at  least 
not  later  than  the  day  after  it  has  been  prepared.  Experience  has  shown  me 
that  it  is  then  quite  as  harmless  and  quite  as  efficacious  as  Pasteur's  vaccine." 

In  the  preparation  of  an  attenuated  virus  by  this  method  Chauveau 
insists  upon  attention  to  the  following  points : 

' '  The  first  rule  to  follow,  and  the  principal  one,  is  to  practise  the  heating 
in  such  a  manner  that  all  parts  of  the  anthrax  blood  are  raised  to  the  re- 
quired temperature  and  withdrawn  from  it  at  the  same  instant.  When  the 
quantity  of  blood  to  be  transformed  to  a  vaccine  is  too  great,  all  parts  are 
not  uniformly  acted  upon  by  the  very  short  exposure  to  heat ;  the  virulent 
agents  in  the  deeper  layers  may,  in  that  case,  preserve  all  of  their  activity, 
and  cause  a  fatal  infection.  To  avoid  this  it  is  best  to  enclose  the  blood  in 
little  cylindrical  pipettes,  1  mm.  in  diameter.  The  extremity  of  these 
pipettes  is  sealed,  and  the  portion  which  contains  the  blood  is  immersed  in  a 
considerable  quantity  of  water  maintained  at  the  proper  temperature.  At 
the  end  of  the  proper  time  they  are  taken  from  the  hot  bath  and  plunged  into 
cold  water. 

' '  Another  rule  should  be  rigorously  observed  if  one  wishes  to  be  sure  of 
success.  The  blood  should  be  collected  under  conditions  which  make  it  sure 
that  the  virulent  agents  introduced  into  the  tubes  all  have  the  same  vitality, 
the  same  activity,  and  that  they  are  impressed  in  the  same  degree  by  the 
heating.  This  is  the  case  when  we  take  the  blood  from  a  guinea-pig  just 
dead,  after  having  survived  from  thirty-six  to  forty-eight  hours  an  inocula- 
tion with  very  active  virus.  Before  introducing  the  blood  into  the  pipettes 
it  should  be  allowed  to  coagulate,  and  the  coagula  should  be  broken  and 
crushed  in  order  to  obtain  a  defibrinated  blood,  which  is  always  very  rich  in 
virulent  bacilli." 

In  a  subsequent  communication  (February  26th,  1883),  Chauveau 
admits  that  the  application  of  this  method  is  somewhat  difficult  and 
delicate  when  blood  is  employed,  and  states  that  it  is  far  more  satis- 
factory to  use  pure  cultures,  which  may  be  attenuated  in  the  same 
way.  He  prefers  to  cultivate  the  bacillus  in  a  bouillon  made  from 
the  flesh  of  a  chicken,  and  to  start  his  culture  by  adding  to  this  bouil- 
lon a  drop  of  blood  from  an  animal  just  dead  from  anthrax.  The  cul- 
ture is  left  for  twenty  hours  in  an  incubating  oven  at  a  temperature  of 
43°  0.  During  this  time  there  is  an  abundant  development  of  the 
bacillus,  and  the  culture  is  ready  to  be  subjected  to  the  attenuating 
action  of  a  higher  temperature.  This  is  accomplished  by  exposure 
to  a  temperature  of  47°  C.  for  a  period  of  one,  two,  three,  or  four 
hours,  according  to  the  degree  of  attenuation  desired.  After  three 
hours'  exposure  the  attenuated  culture  no  longer  kills  guinea-pigs. 
In  a  later  communication  (March  5th,  1883)  Chauveau  states  that  he 
has  ascertained  by  experiment  that  the  degree  of  attenuation  produced 
by  this  method  is  maintained  in  subsequuent  cultures  made  at  43°  C., 
from  the  attenuated  culture  thus  obtained. 

Another  method  of  attenuating  the  virulence  of  anthrax  cultures  is 


278  PROTECTIVE   INOCULATIONS. 

that  described  by  Cliauveau,  in  1885.  Tliis  consists  in  cultivating  the 
bacillus  at  a  temperature  of  38°  to  39°  C.,  under  a  pressure  of  eight 
atmospheres.  Cultures  treated  in  this  way  killed  guinea-pigs,  but 
did  not  kill  sheep,  cattle,  or  horses,  and  constituted  a  suitable  atten- 
uated virus  for  protective  inoculations  in  these  animals.  One  drop 
was  used  for  a  sheep,  and  two  drops  for  a  cow  or  a  horse,  and  the 
immunity  was  proved  to  last  for  a  year. 

Kitt,  in  experiments  made  in  1884  and  1885,  found  that  an  atten- 
uation of  the  virulence  of  anthrax  bacilli  may  be  effected  by  passing 
them  through  birds,  which  have  but  little  susceptibility  to  anthrax 
infection;  but  the  results  obtained  were  not  uniform,  and  the  method 
was  not  thought  to  have  any  great  practical  value.  In  the  same  paper 
Kitt  gives  an  account  of  his  experiments  with  Pasteur's  vaccine,  No. 
1  and  No.  2,  which  he  obtained  from  the  agent  in  Paris.  These  ex- 
periments led  him  to  the  conclusion  that  the  attenuated  cultures  used 
by  Pasteur  are  too  weak.  But  by  passing  them  through  guinea-pigs 
their  virulence  was  increased  so  that  they  served  to  protect  cattle  and 
sheep,  although  not  without  danger  for  the  last-mentioned  animals. 

During  the  year  1882  Pasteur's  method  was  extensively  practised 
in  the  department  of  Eure-et-Loir,  where  anthrax  was  very  prevalent 
and  had  been  the  cause  of  extensive  losses.  The  results  of  these  pro- 
tective inoculations  were  reported  to  the  Academy  of  Sciences  (seance 
of  December  18th,  1882)  by  Pasteur,  who  submitted,  with  some  re- 
marks, a  report  prepared  by  M.  Boutet,  from  which  we  quote  as 
follows : 

"The  number  of  sheep  vaccinated  during1  the  year  has  been  79,392; 
among1  these  flocks  the  average  annual  loss  during1  the  past  ten  years  was 
7,237 — 9.01  percent.  Since  the  vaccinations  but  518  animals  have  died — 0.65 
per  cent.  We  must  observe  that  this  year,  probably  011  account  of  the  great 
humidity,  the  mortality  in  Eure-et-Loir  has  only  been  three  per  cent.  The 
losses  should  therefore  have  been  2,382,  instead  of  518,  without  the  vaccina- 
tions. In  the  flocks  which  were  only  partly  vaccinated  we  had  2,308  vacci- 
nated and  1,659  not  vaccinated  ;  the  loss  among1  the  first  was  8,  or  0.4  per 
cent.;  among1  the  second  the  loss  was  60,  or  3.9  per  cent.  We  call  attention 
to  the  fact  that  in  these  flocks,  in  different  cantons  of  the  department,  the 
sheep  vaccinated  and  not  vaccinated  were  subjected  to  the  same  conditions 
of  soil,  of  lodging,  of  food,  of  temperature,  and  that  consequently  they  were 
exposed  to  identical  influences. 

"The  veterinary  surgeons  in  Eure-et-Loir  have  vaccinated  4,562  animals 
of  the  bovine  species.  Out  of  this  number  the  annual  loss  had  been  322. 
Since  the  vaccinations  only  11  cows  have  died.  That  is,  the  annual  mortality 
has  been  reduced  from  7.03  per  cent,  to  0.24  per  cent. 

"  Some  engorgements,  generally  not  serious,  having  occurred  after  vacci- 
nating1 horses,  and  the  mortality  not  being1  great  in  this  species,  the  veterina- 
rians have  thought  it  prudent  not  to  vaccinate  horses  011  a  large  scale.  Only 
524  were  vaccinated ;  three  of  these  died  after  the  first  vaccination." 

Notwithstanding  this  favorable  report  some  bacteriologists,  and 


PROTECTIVE   INOCULATIONS.  279 

notably  Koch,  were  not  disposed  to  admit  the  practical  value  of  Pas- 
teur's anthrax  inoculations.  At  the  conclusion  of  an  elaborate  me- 
moir published  in  the  second  volume  of  the  "  Mittheilungen  "  of  the 
Imperial  Board  of  Health  of  Germany  (1884),  Koch  and  his  collab- 
orators (Gaff k  v  and  Loffler)  say  : 

"As  now  a  certain  immunity  against  inoculated  anthrax  cannot  be  ob- 
tained by  the  method  of  Pasteur,  as  we  have  seen,  without  considerable 
losses,  and  as  the  immunity  secured  at  the  expense  of  considerable  loss  is 
only  an  imperfect  protection  against  contracting  anthrax  in  the  ordinary 
way,  we  must  consider  the  protective  inoculations  heretofore  practised  as  of 
doubtful  utility,  especially  when  we  remember  that  the  second  inoculation 
with  a  yet  stronger  virus  causes  the  death  of  more  animals  which  may  serve 
to  further  spread  the  disease." 

The  attenuating  influence  of  light  on  the  anthrax  bacillus  and  the 
fact  that  cultures  attenuated  in  this  way  may  be  used  for  protective 
inoculations  was  first  ascertained  by  Arloing  (1886).  Eoux  subse- 
quently (1887)  showed  that  the  presence  of  oxygen  is  a  necessary  fac- 
tor in  the  sterilization  of  cultures  by  exposure  to  sunlight.  Behring, 
wJio  has  since  been  so  active  in  the  field  of  research  to  which  the 
present  volume  relates,  published  an  article  in  the  Centralbkitt  fiir 
klinische  Medicin  in  1888  (September  22d)  in  which  he  attempted  to 
explain  the  natural  immunity  of  white  rats  against  anthrax  infection. 
His  conclusions  are  given  as  follows : 

"1.  The  blood-serum  of  white  rats  is  not  a  favorable  medium  for  the 
anthrax  bacillus." 

"2.  The  blood-serum  of  rats  differs  from  that  of  animals  susceptible  to 
infection  by  its  gi'eater  alkalinity." 

"3.  By  the  addition  of  an  acid  to  the  blood -serum  of  rats  this  becomes  a 
favorable  medium  for  the  growth  of  the  anthrax  bacillus." 

"4.  The  blood-serum  of  rats  which  are  treated,  during  life,  in  such  a  way 
as  to  reduce  the  alkalinity  of  the  blood  becomes  a  suitable  medium  for  the 
development  of  the  anthrax  bacillus." 

As  we  have  pointed  out  in  the  chapter  on  Natural  Immunity,  the 
true  explanation  of  the  facts  ascertained  in  Behring's  experiments  is 
probably  to  be  found,  not  in  the  germiciclal  power  of  the  compar- 
atively small  amount  of  alkali  present  in  the  rat's  serum,  but  in  the 
fact  that  the  germicidal  proteid  produced  by  the  leucocytes  is  only 
soluble  in  an  alkaline  medium.  In  a  paper  published  in  the  Annals 
of  the  Pasteur  Institute  (August,  1888),  Roux  and  Chamberland  have 
given  an  account  of  experiments  made  by  them  which  establish  the 
fact  that  immunity  against  anthrax  maj"  be  established  by  inoculating 
susceptible  animals  with  blood  from  an  animal  dead  from  anthrax,  in 
which  the  anthrax  bacilli  had  been  killed  by  heat  or  removed  by  fil- 
tration (Sur  1'immunite  centre  le  charbon  conferee  par  des  substances 


280  PROTECTIVE    INOCULATIONS. 

chimiques) .     These  experiments  were    commenced  in    1881.     The 
authors  named  say : 

"  In  repeating  the  experiments  of  Toussaint  upon  anthrax  blood  which 
had  been  heated,  we  made  several  observations  which  convinced  us  that  it  is 
possible  to  confer  immunity  against  anthrax  upon  sheep  by  injecting  under 
their  skin  anthrax  blood  which  does  not  contain  any  living  bacilli." 

While  immunity  was  produced  in  this  way,  Roux  and  Chamber- 
land  remark  that  the  sheep  which  had  received  a  comparatively  large 
dose  were  quite  sick  when  subsequently  inoculated  with  a  virulent 
culture,  and  the  immunity  acquired  was  less  reliable  than  that  ob- 
tained by  Pasteur's  method  with  two  vaccines  of  different  degrees  of 
attenuation. 

In  an  investigation  made  by  Hankin,  in  the  laboratory  of  Professor 
Koch  at  the  Hygienic  Institute  of  Berlin,  the  results  of  which  are  given 
in  a  preliminary  account  published  in  the  British  Medical  Journal 
(October  12th,  1889),  the  important  fact  was  ascertained  that  immunity 
may  be  produced  in  susceptible  animals  by  inoculating  them  with  an 
"  albumose  "  isolated  from  anthrax  cultures.  Hankin  gives  the  fol- 
lowing account  of  his  method  of  obtaining  this  immunizing  proteid 
from  anthrax  cultures : 

"  In  the  course  of  my  process  of  preparation  it  is  precipitated  from  its  so- 
lution by  the  addition  of  a  large  bulk  of  absolute  alcohol,  and  well  .washed 
in  this  liquid  to  free  it  from  ptomaines  ;  it  is  well  known  that  all  such  sub- 
stances are  soluble  in  alcohol.  It  is  then  filtered  off  and  dried  ;  then  it  is  re- 
dissolved  and  filtered  through  a  .Chamberland  filter.  A  rough  estimate  of 
the  percentage  of  albumose  present  in  the  clear  solution  thus  obtained  is 
made  colorimetrically  by  means  of  the  biuret  reaction  and  a  peptone  solution 
of  known  strength." 

"In  one  experiment  four  rabbits  (Nos.  23  to  26)  were  inoculated  subcuta- 
neously  with  virulent  anthrax  spores.  No.  26  served  as  a  control  and  died 
in  about  forty  hours.  The  other  three  rabbits  had  the  albumose  solution  in- 
jected into  the  ear-vein  at  the  same  time.  Nos.  24  and  25  each  had  about 
the  five-millionth  of  their  body-weight,  while  No.  23  had  only  the  ten- mil- 
lionth of  its  body-weight  of  albumose.  No.  25  died  in  less  than  forty-eight 
hours,  but  Nos.  23  and  24  survived.  Ten  days  later  Professor  Koch  kindly 
reinoculated  these  two  rabbits  for  me  with  very  virulent  anthrax  from  an 
agar-agar  culture.  Their  temperature  has  remained  normal  since  then,  and 
they  are  now  alive  and  well  a  fortnight  after  this  operation.  I  have  also  suc- 
ceeded in  producing  immunity  in  mice  against  attenuated  anthrax." 

In  a  paper  published  in  the  Proceedings  of  the  Koyal  Society  in 
1890,  Dr.  Sidney  Martin  has  given  an  account  of  his  researches  relat- 
ing to  "  The  Chemical  Products  of  the  Growth  of  Bacillus  Anthracis, 
and  their  Physiological  Action.7'  In  his  experiments  the  cultures 
were  maintained  for  from  ten  to  fifteen  days,  and  the  bacilli  were  then 
removed  by  filtering  through  a  Chamberland  filter.  The  filtrate  was 
found  to  contain : 


PROTECTIVE   INOCULATIONS. 

"  1.  Proto-albumose,  deutero-albumose,  and  a  trace  of  peptone,  all  with 
the  same  chemical  reactions  as  the  similar  bodies  formed  in  peptic  digestion. 
2.  An  alkaloid.  3.  Small  quantities  of  leuciii  or  tyrosin.  The  chief  char- 
acteristic of  the  proto-  and  deutero-albumose  obtained  from  anthrax  cultures 
was  found  to  be  their  strong  alkalinity  in  solution.  This  was  not  removed 
by  prolonged  dialysis  or  by  washing  in  alcohol,  chloroform,  benzene,  or  ether. 
These  proteids  are  precipitated  in  an  alkaline  condition  by  saturation  with 
NaCl  (proto-albumose)  or  (NH4)2SC>4." 

The  alkaloid  found  was  soluble  in  water  or  in  absolute  alcohol, 
was  strongly  alkaline  in  solution,  and  readily  formed  salts  with  acids. 
It  was  slightly  volatile  and  lost  its  poisonous  properties  to  a  great  ex- 
tent when  exposed  to  the  air  for  some  time.  A  mixture  of  the  two 
albumoses  was  toxic,  and  when  injected  into  mice  in  small  amounts 
caused  a  local  subcutaneous  oedema  ending  in  recovery.  Larger 
doses  caused  more  extensive  oedema  and  death.  A  fatal  dose  for  a 
mouse  weighing  twenty-two  grammes  was  0.3  gramme.  Boiling  for  a 
short  time  diminished  the  toxicity  of  these  proteids  without  com- 
pletely destroying  it.  The  alkaloid  produced  similar  symptoms 
when  injected  into  mice,  but  more  promptly  and  in  a  smaller  dose — 0.1 
to  0.15  gramme  killed  a  mouse  weighing  twenty-two  grammes  in  two 
or  three  hours.  Hankin  and  Westbrook  have  more  recently  (1892) 
made  researches  with  reference  to  the  proteids  present  in  anthrax 
cultures.  To  obtain  an  immunizing  albumose  they  cultivated  the 
bacillus  at  20°  C.  in  flesh-extract  solution  (1:1,000)  to  which  fibrin 
was  added.  At  the  end  of  eight  days  a  considerable  precipitate  was 
obtained  by  means  of  ammonium  sulphate.  This  was  placed  in  a 
dialyzer  in  running  water  at  42°  to  45°  C. ;  then  precipitated  by  alco- 
hol and  dissolved  in  a  small  quantity  of  water  (thirty  cubic  centime- 
tres)— five  hundred  cubic  centimetres  of  flesh  extract  treated  in  this 
way  gave  only  0.44  gramme  of  albumose.  Experiments  on  mice 
gave  some  evidence  of  the  immunizing  action  of  this  albumose,  but 
the  results  were  apparently  not  so  definite  as  those  previously  re- 
ported by  Hankin.  Nor  are  the  experiments  of  Petermann,  who 
followed  Hankin 's  method  (1892),  more  satisfactory.  Arloing  ob- 
tained more  favorable  results  by  using  culture  liquids  from  which 
the  bacilli  had  been  removed  by  sedimentation.  A  considerable 
precipitate  was  obtained  when  alcohol  was  added  to  the  culture 
liquid,  but  it  was  found  that  this  precipitate  had  no  immunizing  effect. 
On  the  contrary,  there  remained  in  solution  an  immunizing  sub- 
stance. This  was  obtained  in  a  concentrated  form  by  evaporating  at 
50°  C.  in  a  partial  vacuum.  Experiments  upon  lambs  showed  the 
protective  power  of  this  extract,  and  of  the  culture  liquids  before 
treatment  when  injected  in  considerable  quantity. 


PROTECTIVE   INOCULATIONS. 

In  a  paper  published  in  the  Fortschritte  der  Medicin,  Wysokowicz 
gives  a  resume  of  the  results  obtained  in  Russia  in  protective  inocula- 
tions made  up  to  date  of  publication  (January,  1889).  According  to 
the  author  named,  Professor  Cenkowski,  who  had  made  himself 
familiar  with  Pasteur's  method  while  on  a  visit  to  Paris,  was  the  first 
to  employ  it  in  Russia  (1883).  But  he  found  its  application  to  be 
attended  with  some  difficulties.  The  cultures  attenuated  as  directed 
by  Pasteur  at  42°  to  43°  C.  "showed  a  very  different  degree  of  viru- 
lence in  different  experiments,  and  their  virulence  was  also  changed 
by  keeping."  Experiments  were  therefore  made  with  a  view  to  secur- 
ing a  more  satisfactory  vaccine.  In  an  experiment  made  in  1885, 
1,333  sheep  were  inoculated;  of  these  21  died  from  the  first  inocula- 
tion and  4  from  the  second  (1.86  per  cent).  Subsequently  better 
results  were  obtained,  and  up  to  the  end  of  1888,  20,310  sheep  had 
been  inoculated,  with  an  average  mortality  of  0.87  per  cent  as  a  re- 
sult of  the  inoculations. 

Professor  Cenkowski  found  that  greater  losses  occurred  when  the 
inoculations  were  made  in  midsummer  or  midwinter  than  when  they 
were  made  in  the  spring  or  autumn.  The  losses  from  anthrax  dimin- 
ished among  the  flocks  in  which  the  protective  inoculations  were  prac- 
tised in  proportion  to  the  number  of  sheep  inoculated,  falling  from 
8.3  per  cent  in  1884,  the  year  before  the  inoculations  were  com- 
menced, to  0.13  per  cent  in  1888.  The  author  of  the  paper  states 
that  in  some  parts  of  Russia  the  annual  loss  among  the  sheep  from 
anthrax  is  as  high  as  33  per  cent. 

The  reliability  of  the  protective  inoculations  was  tested  by  a  com- 
mission, to  which  Wysokowicz  belonged.  Fifty  sheep  which  had  been 
inoculated  from  two  to  four  months  previously  were  infected  with  viru- 
lent anthrax  material.  Of  these  only  one  died.  Later,  twenty  sheep 
which  had  been  inoculated  thirteen  months  before  were  inoculated  with 
virulent  material.  Of  these  two  died.  These  favorable  results  are 
ascribed  by  Wysokowicz  to  the  improved  method  of  attenuating 
anthrax  virus  adopted  by  Professor  Cenkowski.  As  a  first  vaccine 
he  employed  a  culture  which  was  stronger  than  that  of  Pasteur,  and 
which  killed  mice  and  caused  the  death  of  one-third  of  the  Ziesel- 
mause  (Spermophilus  citillus)  inoculated.  He  used  as  a  vaccine  an 
attenuated  culture  which  had  been  carried  through  a  series  of  the 
animals  last  mentioned.  His  vaccine,  consisting  of  a  bouillon  cul- 
ture from  a  drop  of  blood  of  the  animal,  was  preserved  by  the  addi- 
tion of  two  parts  of  a  thirty -per-cent  solution  of  pure  glycerin  to 
one  part  of  the  culture. 

For  inoculating  a  sheep  of  average  size  he  used  0.1  to  0.2  cubic 


PROTECTIVE   INOCULATIONS. 

centimetre  of  this  first  vaccine;  for  a  larger  animal,  from  0.3  to  0.5 
cubic  centimetre.  The  second  inoculation  was  made  twelve  days 
after  the  first,  with  a  virus  which  killed  three-fourths  of  the  Ziesel- 
mause  and  from  one-third  to  one-half  of  the  rabbits  inoculated  with  it. 
Numerous  experiments  convinced  Cenkowski  that  no  change  occurred 
in  the  virulence  of  his  different  vaccines  when  they  were  carried 
through  a  series  of  mice  or  of  earless  marmots  (Zieselmause) . 

Hess  reports  that  the  anthrax  inoculations  made  by  Chauveau's 
method  in  the  Canton  Bern,  during  the  years  1886,  1887,  and  1888, 
were  not  attended  with  any  losses  either  from  the  inoculations  or  from 
subsequent  attacks  of  anthrax  among  the  inoculated  animals  (cattle?). 
In  all,  two  hundred  and  fifty-three  animals  were  inoculated  during  the 
three  years  specified. 

Hutyra  (1890)  has  reported  upon  anthrax  inoculations  by  Pasteur's 
method,  as  carried  out  under  the  regulations  of  the  Government  in 
1889.  The  number  of  horses  inoculated  was  130,  2  of  which  died  of 
anthrax  at  a  later  date — not  as  a  result  of  the  inoculation.  This  gives 
a  percentage  of  loss  of  1.35,  which  is  much  below  the  usual  rate  with- 
out protective  inoculations.  Three  thousand  two  hundred  and  sev- 
enty-nine cattle,  belonging  to  32  different  estates,  were  inoculated. 
Of  these  11  died  from  anthrax,  and  2  of  these  as  a  result  of  the  first 
inoculation.  Deducting  these  2  the  loss  was  0.27  per  cent,  whereas 
in  former  years  the  losses  in  the  same  herds  had  been  from  6  to 
12  per  cent.  Twenty -two  thousand  seven  hundred  and  sixty- 
seven  sheep  were  inoculated  on  23  different  estates.  One  hundred 
and  sixty-two  of  these  died  from  the  first  inoculation  and  59  within 
twelve  days  after  the  second  inoculation.  In  the  course  of  the  year 
432  of  the  inoculated  animals  died  from  anthrax — in  all  a  loss  of 
2.18  per  cent.  In  the  absence  of  protective  inoculations  the  annual 
loss  in  these  flocks  had  been  about  10  per  cent.  It  was  found  that 
lambs  four  months  old  could  be  inoculated  with  the  same  dose  as  the 
older  sheep,  and  without  any  greater  loss  as  a  result  of  the  operation. 

The  result  of  anthrax  inoculations  made  in  France  by  Pasteur's 
method  during  the  twelve  years  ending  in  1894  have  been  sum- 
marized by  Chamberland.  The  veterinarians  who  made  the  inocula- 
tions were  each  year  called  upon  to  answer  the  following  questions : 
1.  Number  of  animals  inoculated.  2.  Number  of  deaths  from  first 
inoculation.  3.  Number  of  animals  dying  within  twelve  days  after 
second  inoculation.  4.  Number  of  animals  dying  of  anthrax  within 
a  year  after  protective  inoculations.  5.  The  yearly  average  loss 
before  inoculations  were  practised.  The  total  number  of  animals 
inoculated  during  the  period  to  which  this  report  refers  was  1,788,- 


284  PROTECTIVE    INOCULATIONS. 

677  sheep  and  200,962  cattle.  Tlie  average  annual  loss  before 
these  protective  inoculations  were  practised  is  said  to  have  been 
about  ten  per  cent  for  sheep  and  five  per  cent  for  cattle.  The  total 
mortality  from  this  disease  among  inoculated  animals,  including  that 
resulting  from  the  inoculations,  was  0.94  per  cent  for  sheep  and  0.34 
per  cent  for  cattle.  Chamberland  estimates  that  the  total  saving  as  a 
result  of  the  inoculations  practised  has  been  5,000,000  francs  for 
sheep  and  2,000,000  francs  for  cattle. 

Podmolinoff  gives  the  following  summary  of  results  obtained  in 
1892  and  1893  in  the  "  government  of  Cherson  "  (Austria) :  Number 
of  sheep  inoculated,  67,176;  loss,  294  =  0.43  per  cent.  Number  of 
horses  inoculated,  1,452;  loss,  8.  Number  of  cattle  inoculated, 
3,652;  loss,  2.  The  conclusion  is  reached  that  Pasteur's  method  of 
inoculation  affords  an  immunity  against  infection  with  virulent  an- 
thrax bacilli  in  greater  amounts  than  could  ever  occur  under  natu- 
ral conditions. 

BUBONIC    PLAGUE. 

A  number  of  prominent  bacteriologists  have  been  engaged  in  re- 
searches relating  to  the  prevention  and  cure  of  bubonic  plague  by 
means  of  an  antitoxic  serum,  obtained  by  the  same  method  and  in 
accordance  with  the  same  fundamental  scientific  principle  as  in  the 
case  of  the  antitoxic  serum  which  is  now  so  successfully  employed  in 
the  treatment  of  diphtheria.  The  experiments  thus  far  made  have 
apparently  been  attended  with  a  considerable  degree  of  success.  Pro- 
fessor Calmette  reports  that  the  serum  of  Yersin  prepared  at  the  Pas- 
teur Institute  in  Paris  proved  to  be  curative  in  a  considerable  propor- 
tion of  the  cases  treated  during  the  recent  outbreak  at  Oporto,  and  that 
protective  inoculation  conferred  a  temporary  immunity,  which,  how- 
ever, did  not  last  longer  than  twenty  days.  The  mortality  in  cases 
not  treated  by  Yersin 's  serum  was  70  per  cent,  in  those  treated  with 
it  13  per  cent. 

The  inoculations  made  by  Haffkine  in  Bombay  appear  to  have  been 
quite  successful.  In  his  first  experiment  8,142  persons  were  inocu- 
lated. Of  these  18  subsequently  contracted  the  disease  and  2  died. 
Among  4,926  persons  inoculated  a  single  time  at  Dharwan,  45  were 
subsequently  attacked  and  15  died;  while  among  3,387  persons  in 
whom  a  second  inoculation  was  made,  only  2  were  attacked.  Haff- 
kine uses  in  his  inoculations  a  sterilized  culture  of  the  plague  bacil- 
lus. The  inoculation  is  followed  by  slight  fever  and  enlargement  of 
the  nearest  lymphatic  glands.  All  symptoms  disappear  at  the  end 
of  two  or  three  da  vs. 


PROTECTIVE   INOCULATIONS.  285 

The  duration  of  the  immunity  resulting  from  these  inoculations 
has  not  been  definitely  determined,  although  in  a  majority  of  those 
inoculated  it  appears  to  have  afforded  protection  for  at  least  five  or 
six  months.  Haffkine's  method  of  preparing  his  material  for  protec- 
tive inoculations  is  as  follows:  A  kilogramme  of  finely  chopped  goat's 
flesh  is  macerated  in  diluted  hydrochloric  acid,  and  then  placed  in  an 
autoclave  and  heated  for  six  hours  under  a  pressure  of  three  atmos- 
pheres. This  is  filtered,  neutralized  with  KOH,  and  diluted  up  to 
three  litres.  The  plague  bacillus  is  grown  in  this  medium.  Accord- 
ing to  Haffkine,  when  the  bacillus  is  planted  upon  the  surface  of  this 
medium,  a  characteristic  growth  results.  If  undisturbed  for  five  or 
six  days  delicate  thread-like  processes  are  seen  hanging  in  the  culture 
medium  resembling  stalactites  suspended  from  the  roof  of  a  cavern. 
This  growth  is  said  to  be  peculiar  to  the  plague  bacillus.  To  make 
the  prophylactic  the  bacillus  is  grown  in  a  darkened  room  in  large 
flasks.  In  India  it  is  unnecessary  to  use  a  thermostat.  Five  or  six 
crops  of  the  stalactites  are  growrn  and  shaken  to  the  bottom  of  the 
flasks.  This  takes  about  six  weeks.  The  culture  is  then  sterilized 
in  a  water  bath  at  70°  C.,  the  time  required  beiug  about  three  hours. 
A  little  carbolic  acid  or  thymol  is  then  added,  and  the  material,  after 
shaking  to  distribute  the  bacteria,  is  decanted  into  small  bottles.  It 
is  now  ready  for  use,  and  is  usually  injected  into  the  subcutaneous 
connective  tissue  in  doses  of  two  cubic  centimetres.  A  second  inocu- 
lation in  from  fourteen  to  twenty  days  is  recommended  by  Leumaun, 
and  after  this  the  blood  of  the  inoculated  individual  usually  gives  the 
Widal  reaction. 

CHICKEN   CHOLERA. 

Pasteur's  researches  with  reference  to  the  etiology  of  the  disease 
known  in  France  as  cholera  des  ponies  first  led  him  to  the  discovery 
that  a  virulent  culture  of  a  pathogenic  bacterium  may  become  "  atten- 
uated "  by  certain  agencies,  and  that  immunity  may  be  conferred  upon 
susceptible  animals  by  inoculating  them  with  such  attenuated  culture. 
We  now  know  that  his  microbe  of  fowl  cholera  is  a  widely  distributed 
bacillus,  which  is  frequently  encountered  in  putrefying  material,  and 
that  it  is  also  extremely  fatal  to  pigeons,  pheasants,  sparrows,  rabbits, 
and  mice.  Also  that  the  same  or  nearly  allied  species  may  produce 
an  infectious  disease  of  swine  (Schweineseuche),  of  cattle  (Hinder- 
seuche),  and  of  deer  ( Wildseuche). 

Subcutaneous  injection  of  a  minute  quantity  of  a  virulent  culture 
usually  kills  chickens  within  forty-eight  hours.  Some  time  before 
death  the  fowl  falls  into  a  somnolent  condition,  and,  with  drooping 


286  PROTECTIVE  INOCULATIONS. 

wings  and  ruffled  feathers,  remains  standing  in  one  place  until  it  dies. 
Infection  may  also  occur  from  the  ingestion  of  food  moistened  with  a 
culture  of  the  bacillus  or  soiled  with  the  discharges  from  the  bowels 
of  other  infected  fowls.  At  the  autopsy  the  mucous  membrane  of  the 
small  intestine  is  found  to  be  inflamed  and  studded  with  small  hem- 
orrhagic  foci,  as  are  also  the  serous  membranes ;  the  spleen  is  notably 
enlarged.  The  bacilli  are  found  in  great  numbers  in  the  blood,  in  the 
various  organs,  and  in  the  contents  of  the  intestine.  In  rabbits  death 
commonly  occurs  in  from  sixteen  to  twenty  hours,  and  is  often  pre- 
ceded by  convulsions.  The  temperature  is  elevated  at  first,  but 
shortly  before  death  it  is  reduced  below  the  normal.  The  post-mor- 
tem appearances  are :  swelling  of  the  spleen  and  lymphatic  glands ; 
ecchymoses  or  diffuse  hemorrhagic  infiltrations  of  the  mucous  mem- 
branes of  the  digestive  and  respiratory  passages,  and  in  the 
muscles ;  and  at  the  point  of  inoculation  a  slight  amount  of  inflamma- 
tory oedema.  The  bacilli  are  found  in  considerable  numbers  in  the 
blood  within  the  vessels,  or  in  that  which  has  escaped  into  the  tissues 
by  the  rupture  of  small  veins.  They  are  not,  however,  so  numerous 
as  in  some  other  forms  of  septicaemia— e.g.,  anthrax,  mouse  septi- 
caemia— when  an  examination  is  made  immediately  after  death ;  later, 
the  number  may  be  greatly  increased  as  a  result  of  post-mortem  mul- 
tiplication within  the  vessels.  The  rabbit  is  so  extremely  susceptible 
to  infection  by  this  bacillus  that  inoculation  in  the  cornea  by  a  slight 
superficial  wound  usually  gives  rise  to  general  infection  and  death. 
This  animal  may  also  be  infected  by  the  ingestion  of  food  contami- 
nated with  a  culture  of  the  bacillus.  It  is  by  this  means  that  Pasteur 
proposed  to  destroy  the  rabbits  in  Australia,  which  have  multiplied 
in  that  county  to  such  an  extent  as  to  constitute  a  veritable  pest. 
Both  in  fowls  and  in  rabbits  the  disease  may,  under  certain  circum- 
stances, run  a  more  protracted  course — e.</.,  when  they  are  inoculated 
with  a  small  quantity  of  an  attenuated  culture.  In  less  susceptible 
animals — guinea-pigs,  sheep,  dogs,  horses — a  local  abscess,  without 
general  infection,  may  result  from  the  subcutaneous  injection  of  the 
bacillus ;  but  these  animals  are  not  entirely  immune.  In  the  infec- 
tious maladies  of  swine,  cattle,  deer,  and  other  large  animals,  to 
which  reference  has  been  made,  and  which  are  believed  to  be  due  to 
the  same  bacillus,  the  symptoms  and  pathological  appearances  do  not 
entirely  correspond  with  those  in  the  rabbit  or  the  fowl ;  but  the  ba- 
cillus as  obtained  from  the  blood  of  such  animals  corresponds  in  its 
morphological  and  biological  characters  with  Pasteur's  microbe  of 
fowl  cholera,  and  Koch's  bacillus  of  rabbit  septicaemia,  and  pure 
cultures  from  the  various  sources  mentioned  are  equally  fatal  to  rab- 


PROTECTIVE   INOCULATIONS.  287 

bits  and  to  fowls.  lu  the  larger  animals  pulmonary  and  intestinal 
lesions  are  developed,  and  in  swine  a  diffused  red  color  of  the  skin, 
similar  to  that  observed  in  the  disease  known  in  Germany  as  Schicein- 
erothlauf  (Fr.  rouget)  is  sometimes  seen. 

According  to  Baumgarten,  bacilli  from  JVildseiiche  or  from  Rinder- 
seaclie  inoculated  into  swine  give  rise  to  fatal  Schweineseuche,  and 
bacilli  from  any  of  these  forms  of  disease,  when  inoculated  into  pig- 
eons, produce  characteristic  fowl  cholera ;  but  the  bacillus  as  obtained 
from  Schweinesucheoi  W&dseucke  is  not  fatal  to  chickens,  and  the  ba- 
cillus from  Schweineseuche  is  fatal  to  guinea-pigs,  which  have  but 
slight  susceptibility  to  the  bacillus  of  rabbit  septicaemia.  Notwith- 
standing these  differences,  he  agrees  with  Hueppe  in  the  view  that  the 
bacilli  from  the  various  sources  mentioned  are  specifically  identical; 
although  evidently,  if  this  view  is  adopted,  we  must  admit  that  varie- 
ties exist  which  differ  somewhat  in  their  pathogenic  power. 

In  this  volume  this  bacillus  is  described  under  the  name  Bac'Jlus 
septiccemice  hcemorrhagicce,  first  proposed  for  it  by  Hueppe.  In  the 
present  chapter  we  shall  give  an  account  of  the  experimental  evidence 
relating  to  protective  inoculations  in  various  animals,  with  the  differ- 
ent varieties  of  the  bacillus  in  question  which  have  been  encountered. 

It  seems  probable  that  the  same  bacillus  was  the  cause  of  the  fatal 
form  of  septicaemia  studied  by  Davaine,  which  resulted  from  the  in- 
oculation of  susceptible  animals  with  putrefying  blood.  These  ex- 
periments by  the  distinguished  French  physician  constitute  an  im- 
portant part  of  the  pioneer  work  in  this  field  of  research.  They  were 
commenced  in  1868,  and  are  published  in  the  Bulletin  of  the  Academy 
of  Medicine  (seance  of  Feburary  18th,  1879). 

Davaine,  in  the  paper  referred  to,  calls  attention  to  the  fact,  devel- 
oped by  his  experiments,  that  there  is  a  great  difference  in  the  resist- 
ing power  of  different  animals  to  the  form  of  septicaemia  which  had 
been  the  subject  of  his  investigations.  Thus  the  rabbit  succumbed 
when  inoculated  with  a  millionth  part  of  a  drop  of  blood,  while  guinea- 
pigs  and  dogs  remained  unaffected  by  such  small  doses.  With  refer- 
ence to  the  specific  cause  of  the  form  of  septicaemia  studied  by  him, 
Davaine  says : 

"The  virus  is  one  of  the  bacteria  of  putrefaction.  I  say  'one  of  the  bac- 
teria,' because  there  is  reason  to  believe  that  there  are  among  these  minute 
organisms  numerous  species  which  do  not  all  develop  at  the  same  time  when 
they  are  present  in  various  media." 

Davaine  also  discovered  the  fact  that  infection  depends,  within 
certain  limits,  upon  the  quantity  of  bacteria  introduced  into  the  tis- 
sues. He  says : 


288  PROTECTIVE   INOCULATIONS. 

"This  question  of  quantity  was  manifest  in  our  experiments.  Not  only 
did  it  vary  in  different  species,  the  rabbit  and  the  dog,  for  example,  but  it 
may  vary  in  the  same  species/' 

The  identity  of  "  Davaine's  septicaemia  "  with  Pasteur's  cholera  des 
ponies  is  made  still  more  probable  by  the  experimental  evidence 
offered  by  Toussaint  in  a  communication  to  the  French  Academy  of 
Sciences,  made  by  M.  Bouley  at  the  seance  of  July  25th,  1881.  In 
this  communication  Toussaint  says : 

"Three  years  ago,  July  8th,  1878,  I  had  the  honor  to  present  to  the 
Academy  an  account  of  a  malady  due  to  microbes,  which  I  identified  with 
that  studied  by  Davaine  in  1864  and  1865,  and  which  he  differentiated  from 
anthrax,  for  which  it  had  been  mistaken  by  Leplat  and  Jaillard. 

"In  the  month  of  December,  1878,  I  made  acquaintance  with  fowl 
cholera,  and  already,  in  my  thoughts,  I  identified  this  disease  with  that 
which  I  had  observed  in  my  experiments  made  early  in  the  year.  The  mi- 
crobes of  the  two  diseases  resembled  each  other  perfectly  and  behaved  the 
same  when  inoculated  in  rabbits.  I  had,  even  in  1879,  sent  to  M.  Bouley 
two  notes,  in  which  I  called  attention  to  the  analogies  which  exist  between 
the  parasites  of  the  two  diseases  and  the  lesions  which  they  determine,  not 
only  in  the  rabbit  but  also  in  pigeons  and  fowls. 

"The  experiments  of  the  same  kind  made  at  the  end  of  1879  and  in  1880 
caused  me  to  insert  the  note  published  on  page  301,  vol.  xci.,  of  the  Comptes- 
rendus,  under  the  title  of  '  Identity  of  Acute  Experimental  Septicaemia 
and  Fowl  Cholera.'  I  gave  a  resume  in  this  note  of  five  series  of  experi- 
ments which  had  demonstrated  to  me  that  inoculations  of  the  microbe  of  sep- 
ticaemia give  rise  to  the  manifestations  of  fowl  cholera.  These  results  have 
recently  been  confirmed  by  additional  facts." 

Toussaint  closes  his  paper  by  some  remarks  upon  the  origin  of 
epidemics  of  fowl  cholera,  which  we  quote  because  we  believe  that  the 
additions  made  to  our  knowledge  of  the  microbe  which  causes  this 
disease  give  support  to  the  views  advanced  by  him  in  1881 : 

"  The  causes  which  determine  epidemics  of  fowl  cholera  are  yet  unknown. 
It  has  been  supposed  that  putrefactive  substances  may  give  rise  to  them,  and 
this  has  led  to  the  recommendation  of  cleanliness  and  disinfection  for  their 
prevention.  The  microbe  which  kills  the  first  fowl  in  an  epidemic  certainly 
came  from  some  anterior  generation  which  had  killed  others.  But  how  was 
it  perpetuated  ?  Do  not  the  facts  which  demonstrate  the  development  of  sep- 
ticaemia from  material  undergoing  putrefaction  throw  some  light  on  the  ques- 
tion of  etiology  ?  Is  it  not  probable  that  the  fowls  find  the  conditions  of 
infection  with  cholera  in  the  presence  of  organic  matter  undergoing  putrefac- 
tion, which  may  serve  as  a  culture  medium  for  the  germs  of  septicaemia 
which  are  in  suspension  in  the  air  together  with  the  ordinary  germs  of  putre- 
faction?" 

Pasteur's  first  communication  relating  to  the  etiology  of  fowl 
cholera  was  made  to  the  French  Academy  at  the  seance  of  February 
9th,  1880.  In  this  communication  he  calls  attention  to  the  fact  that 
when  fowls  are  fed  with  bread  or  meat  soiled  with  a  small  quantity 
of  a  culture  of  the  microbe  of  fowl  cholera  they  become  infected  and 
their  discharges  contain  the  bacillus  in  large  numbers,  a  fact  which 


PROTECTIVE   INOCULATIONS.  289 

readily  accounts  for  the  spread  of  the  disease  in  a  poultry  yard  when 
a  case  occurs. 

In  the  same  communication  Pasteur  records  his  observation  that 
"  by  a  certain  change  in  the  method  of  cultivation  the  infectious  mi- 
crobe may  be  caused  to  have  a  diminished  virulence."  Also  the  fact 
that  fowls  inoculated  with  this  "  attenuated  "  virus  recover  and  are 
subsequently  immune  against  infection  by  the  most  virulent  microbes. 
In  concluding  this  communication  Pasteur  says : 

' '  It  appears  to  be  superfluous  to  point  out  the  principal  result  of  the  facts 
which  I  have  had  the  honor  to  present  to  the  Academy.  There  are  two,  how- 
ever, which  it  may  be  useful  to  mention.  These  are,  first,  the  hope  of  ob- 
taining artificial  cultures  of  all  kinds  of  virus  ;  second,  the  idea  of  seeking 
for  virus  vaccines  of  the  virulent  maladies  which  have  devastated  so  often, 
and  still  devastate,  the  human  race,  and  are  such  a  scourge  to  J;hat  branch  of 
agriculture  which  relates  to  the  breeding  of  domestic  animals." 

In  his  communication  of  October  26th,  1880,  Pasteur  gives  his  rea- 
sons for  concluding  that  attenuation  of  virulence  is  due  to  the  action 
upon  the  microbe  of  atmospheric  oxygen.  He  infers  this  from  the 
fact,  demonstrated  by  experiment,  that  when  cultures  are  placed  in 
hermetically  sealed  tubes,  from  which  the  oxygen  present  is  soon  ex- 
hausted by  the  growth  of  the  microbe,  they  do  not  become  attenuated 
in  virulence;  whereas  cultures  which  are  freely  exposed  to  the  air 
gradually  become  attenuated.  Pasteur  sees  in  this  an  important  fact 
bearing  upon  the  explanation  of  the  natural  extinction  of  epidemics. 
He  says : 

' '  May  we  not  suppose,  then,  that  it  is  to  this  influence  that  we  must  at- 
tribute, in  the  present  as  in  the  past,  the  limitation  of  great  epidemics  ? " 

In  his  communication  to  the  French  Academy,  made  on  February 
28th,  1881,  Pasteur  treats  of  the  attenuation  of  virulence  by  the  method 
above  referred  to  and  by  the  method  of  Toussaint,  and  also  of  the  re- 
establishment  of  the  virulence  of  attenuated  cultures.  He  says : 

' '  The  secret  of  the  return  to  virulence  rests  solely,  at  present,  upon  suc- 
cessive cultures  in  the  bodies  of  certain  animals/' 

Thus  he  had  found  by  experiment  that  the  anthrax  bacillus  might 
be  so  attenuated  that  it  was  harmless  for  grown  guinea-p?gs,  or  even 
for  guinea-pigs  a  month  or  a  week  old,  but  it  would  still  kill  guinea- 
pigs  just  born — a  day  old.  By  inoculating  an  older  pig  with  the  blood 
of  this  one,  and  so  on,  the  virulence  was  gradually  augmented,  until 
finally  a  virus  might  be  obtained  which  would  kill  adult  animals,  and 
even  sheep.  In  the  same  way  the  attenuated  microbe  of  fowl  cholera 
could  be  restored  to  virulence  by  first  inoculating  small  birds,  such  as 

sparrows  or  canaries. 
19 


290  PROTECTIVE    INOCULATIONS. 

Applying  these  facts,  demonstrated  by  his  experiments,  to  the  ex- 
planation of  the  origin  of  epidemics,  Pasteur  says : 

"  I  finished  my  communication  on  October  26th  by  calling  attention  to  the 
attenuation  of  viruses  by  exposure  to  the  air  as  being  probably  one  of  the  fac- 
tors in  the  extinction  of  great  epidemics.  The  facts  presented  in  this  paper, 
in  their  turn,  may  serve  to  explain  the  so-called  'spontaneous  development' 
of  these  scourges. 

' '  An  epidemic  which  has  been  extinguished  by  the  attenuation  of  its  virus 
may  be  reborn  by  the  reinforcement  of  this  virus  under  certain  influences. 
The  accounts  which  I  have  read  of  the  spontaneous  appearance  of  the  plague 
appear  to  me  to  offer  examples  of  this  ;  for  example,  the  plague  at  Benghazi, 
in  1856-58,  the  outbreak  of  which  could  not  be  traced.  The  plague  is  a  viru- 
lent malady  which  belongs  to  certain  countries.  In  all  of  these  countries  its 
attenuated  virus  ought  to  exist,  ready  to  resume  its  active  form  when  condi- 
tions as  to  climate,  famine,  and  distress  again  occur.  There  are  other  viru- 
lent maladies  which  appear  '  spontaneously '  in  all  countries  ;  such  as  camp 
typhoid.  Without  doubt  the  germs  of  the  microbes  which  cause  these  last- 
mentioned  maladies  are  everywhere  distributed.  Man  carries  them  upon  him 
or  in  his  intestinal  canal  without  great  damage,  but  ready  to  become  danger- 
ous, when,  owing  to  constipation  or  to  successive  development  upon  the  sur- 
face of  wounds,  in  bodies  enfeebled  or  otherwise,  their  viruleiicy  is  pro- 
gressively reinforced. M 

We  believe  that  the  more  complete  our  knowledge  relating  to  the 
origin  and  extinction  of  epidemics,  of  the  kind  referred  to  by  Pasteur, 
becomes,  the  more  apparent  will  be  the  value  of  his  inductions  and 
the  clearness  of  his  scientific  foresight. 

Toussaint,  on  July  25th,  1881,  reported  the  results  of  his  experi- 
ments upon  protecting  fowls  by  a  "  new  method  of  vaccination."  This 
consisted  in  inoculating  them  with  the  blood  of  a  rabbit  which  had  re- 
cently died  from  septicaemia  produced  by  the  same  microbe.  As  a 
result  of  such  inoculations  the  fowls  had  slight  local  lesions  at  the 
point  of  inoculation,  and  soon  recovered.  They  were  subsequently 
found  to  be  immune.  Cultures  from  the  blood  of  a  septicsemic  rabbit 
were  found  to  act  in  the  same  way.  When  the  culture  had  been  passed 
through  a  pigeon,  and  had  then  killed  a  fowl,  according  to  Toussaint, 
it  preserved  its  virulence  whsn  subsequently  passed  through  the 
rabbit. 

Salmon,  in  the  "  Report  of  the  Commissioner  of  Agriculture  "  for 
1881  and  1882,  gives  an  account  of  his  experiments  in  producing  im- 
munity by  the  use  of  a  diluted  virus.  He  says : 

"The  experiments  of  Chauveau,  taken  with  my  own,  indicate  that  this 
method  is  capable  of  generalization  to  the  same  extent  as  that  discovered  by 
Pasteur :  while  the  ease  and  quickness  with  which  the  vaccine  is  prepared, 
the  certainty  of  effects,  the  economy  of  material,  and  the  more  perfect  pro- 
tection are  points  which  would  appear  to  make  it  decidedly  superior. 
Wherever  the  cholera  of  fowls  is  raging  a  standard  cultivation  may  be  made 
and  the  vaccine  obtained  within  twenty-four  hours  ;  a  single  drop  of  such  a 
cultivation  will  vaccinate  ten,  twenty,  or  even  forty  thousand  fowls,  and 
within  three  weeks  of  the  commencement  of  work  the  most  susceptible  of  our 


PROTECTIVE    INOCULATIONS.  291 

fowls  are  insusceptible  to  inoculation  with  the  strongest  virus.  And  this, 
without  any  sickness,  or  even  local  necroses,  which  Pasteur  describes  as  fol- 
lowing vaccinations  with  his  attenuated  virus." 

In  discussing  the  practical  value  of  this  method  Salmon  estimates 
the  cost  as  trifling—  "  not  more  than  half  a  day's  time  of  one  man  for 
one  hundred  fowls,  even  if  three  inoculations  were  made." 

In  a  paper  on  protective  inoculations  against  fowl  cholera,  by  Kitt, 
in  the  Deutsche  Zeitsclirlft  fur  Tltiermedicm  (December  20th,  1886),  the 
conclusion  is  reached  that  these  inoculations  undoubtedly  protect  the 
fowls  from  infection  either  in  the  natural  way  or  by  inoculations  with 
virulent  material.  But  Kitt  doubts  the  practical  utility  of  the  method 
for  the  arrest  of  epidemics  of  this  disease  in  the  poultry  yard;  and, 
as  we  think  with  justice,  prefers  to  depend  upon  cleanliness,  disin- 
fection, and  prompt  removal  of  infected  fowls.  As  he  points  out,  a 
considerable  time  is  required  to  produce  complete  immunity,  and  two 
inoculations  are  often  insufficient.  Pasteur  had  previously  reported 
that  a  third  inoculation  is  usually  required.  But  the  infection 
spreads  so  rapidly  when  an  epidemic  is  developed  in  a  poultry  yard 
that  a  large  proportion  of  the  fowls  would  be  likely  to  perish  before 
the  protective  inoculations  could  be  carried  out.  Another  objection 
is  that  when  inoculated  in  the  breast  muscle  the  value  of  the  fowl  for 
the  table  is  reduced,  and  when  inoculated  in  the  wing  an  unpleasant- 
looking  scab  is  left  at  the  point  of  inoculation.  The  cost  in  material 
and  time  required  to  carry  out  the  three  successive  inoculations  is 
also  an  objection  to  the  practical  application  of  the  method.  More- 
over, the  excreta  of  the  inoculated  fowls  contain  the  pathogenic  mi- 
crobe, and  it  would  evidently  be  unwise  to  practise  inoculations  in 
poultry  yards  not  already  infected.  Kitt  states  also  that  he  has 
always  succeeded  in  stamping  out  the  disease  very  promptly  by  the 
other  measures  referred  to — disinfection,  cleanliness,  separation  of 
all  fowls  which  show  any  indications  of  being  infected. 

In  a  more  recent  paper  (1893)  Kitt  reports  his  success  in  confer- 
ring immunity  upon  fowls  by  a  new  method,  which  is,  however, 
rather  of  scientific  interest  than  of  practical  value.  He  first  experi- 
mented to  see  whether  the  blood  serum  or  tissue  juices  of  immune 
fowls  would  give  immunity  against  cholera  to  other  fowls,  and  ob- 
tained a  successful  result.  He  was  not,  however,  able  to  produce  im- 
munity in  pigeons  or  in  rabbits  by  the  same  method.  He  next  under- 
took to  determine  whether  the  immunizing  substance  was  present  in 
the  eggs  of  fowls  which  had  an  immunity  as  a  result  of  protective  in- 
oculations. The  albumen  and  yolk  of  the  egg,  in  doses  of  five  to  ten 
cubic  centimetres,  was  injected  into  the  breast  of  fowls,  and  at  the  end 


292  PROTECTIVE   INOCULATIONS. 

of  ten  days  a  second  inoculation  of  the  same  kind  was  made.  Six  days 
after  the  second  inoculation  the  fowls  (five)  and  a  control  hen  were 
inoculated  with  virulent  blood  from  a  pigeon,  and  at  the  same  time  fed 
with  the  chopped-up  flesh  and  liver  of  a  pigeon  just  dead  from  fowl 
cholera.  The  control  hen  died  on  the  following  day  from  typical 
cholera,  the  others  remained  in  perfect  health. 

CHOLERA. 

The  spirillum  discovered  by  Koch  in  1884  is  now  generally  recog- 
nized as  the  specific  cause  of  Asiatic  cholera.  But  recent  researches 
indicate  that  there  are  numerous  pathogenic  varieties  of  this  spirillum, 
and  show  that  either  an  attenuated  cholera  spirillum  or  a  closely  allied 
saprophyte  is  not  infrequently  found  in  the  water  of  rivers  in  various 
parts  of  Europe.  As  this  spirillum  is  found  in  the  intestine  of  cholera 
patients,  and  not  in  the  blood,  it  is  evident  that  its  pathogenic  action 
depends  upon  the  chemical  products  developed  during  its  growth, 
and  this  inference  is  fully  justified  by  the  results  of  experiments  upon 
the  lower  animals.  These  chemical  products  have  been  studied  by 
Brieger,  Pfeiffer,  Scholl,  Gamaleia,  Westbrook,  and  others. 

Brieger  (1887)  succeeded  in  isolating  several  toxic  ptomaines  from 
cultures  of  the  cholera  spirillum,  some  of  which  had  previously  been 
obtained  from  other  sources — cadaverin,  putrescin,  creatinin,  methyl- 
guanidin.  In  addition  to  these  he  obtained  two  toxic  substances  not 
previously  known.  One  of  these  is  a  diamine,  resembling  trimethyl- 
diamine ;  it  gave  rise  to  cramps  and  muscular  tremor  in  inoculated 
animals.  The  other  poison  reduced  the  frequency  of  the  heart's 
action  and  the  temperature  of  the  body  in  the  animals  subjected  to 
experiment.  In  more  recent  researches  made  by  Brieger  and  Frankel 
(1890) ,  a  toxalbumin  was  obtained  from  cholera  cultures  which,  when 
injected  subcutaneously  into  guinea-pigs,  caused  their  death  in  two  or 
three  days,  but  had  no  effect  upon  rabbits. 

Pfeiffer  has  more  recently  (1892)  published  his  extended  researches 
relating  to  the  cholera  poison.  He  finds  that  recent  aerobic  cultures 
of  the  cholera  spirillum  contain  a  specific  toxic  substance  which  is 
fatal  to  guinea-pigs  in  extremely  small  doses.  This  substance  stands 
in  close  relation  to  the  bacterial  cells,  and  is  perhaps  an  integral  part 
of  the  same.  The  spirilla  may  be  killed  by  chloroform,  thymol,  or 
by  desiccation,  without  apparent  injury  to  the  toxic  potency  of  this 
substance.  It  is  destroyed,  however,  by  absolute  alcohol,  by  concen- 
trated solutions  of  neutral  salts,  and  by  the  boiling  temperature,  and 
secondary  toxic  products  are  formed  which  have  a  similar  pathogenic 


PROTECTIVE   INOCULATIONS.  293 

action  but  are  from  ten  to  twenty  times  less  potent.  Similar  toxic 
products  were  obtained  by  Pfeiffer  from  cultures  of  the  Finkler-Prior 
spirillum  and  from  Spirillum  Metchnikovi. 

Sclioll  (1890)  took  advantage  of  the  fact,  previously  demonstrated 
by  Hueppe,  that  cultures  of  the  cholera  spirillum  in  egg  albumen,  in 
the  absence  of  oxygen,  are  more  toxic  than  ordinary  bouillon  cultures. 
Cultures  were  made  by  Hueppe's  method  in  hen's  eggs.  No  poison- 
ous ptomaines  were  found,  but  two  toxic  albuminous  substances  were 
obtained.  The  albuminous  liquid  from  the  egg  cultures  was  dropped 
into  ten  times  its  volume  of  absolute  alcohol,  which  caused  a  white 
precipitate,  a  portion  of  which  sank  to  the  bottom  while  another  por- 
tion floated  on  the  surface.  The  portion  which  floated  was  easily 
dissolved  in  a  very  dilute  solution  of  potash  and  could  be  precipitated 
from  this  solution  by  the  careful  addition  of  acetic  acid,  but  dissolved 
in  an  excess  of  this  acid.  It  dissolved  also  in  a  seven-per-cent  salt 
solution,  but  was  precipitated  by  a  saturated  solution.  It  gave  the 
biuret  and  xanthoprotein  reaction.  This  substance  proved  to  be  very 
poisonous.  It  killed  guinea-pigs  within  twenty  minutes  when  a  few 
cubic  centimetres  of  the  alkaline  solution — potash — were  injected  into 
the  cavity  of  the  abdomen.  Scholl  calls  this  substance  cholera-toxo- 
globulin.  The  precipitate  which  fell  to  the  bottom  of  the  receptacle 
wras  washed  with  alcohol,  then  digested  with  water  for  twenty  minutes 
at  40°  C.  Very  little  was  apparently  dissolved  out  by  this  procedure, 
but  this  little  proved  to  be  very  toxic.  In  from  one  to  three  minutes 
after  the  injection  of  a  few  cubic  centimetres  of  the  solution  into  the 
peritoneal  cavity  of  a  guinea-pig  the  animal  died.  This  aqueous 
solution  gave  the  biuret  and  xanthoprotein  reaction ;  it  was  precipi- 
tated by  mercuric  chlorid,  nitrate  of  mercury,  and  tannin,  but  not  by 
a  saturated  solution  of  ammonium  sulphate  or  acetic  acid.  This  sub- 
stance Scholl  calls  cholera-toxo-pepton.  The  toxic  action  of  these 
substances  is  destroyed  by  a  temperature  of  100°  C.,  maintained  for 
half  an  hour,  or  by  40°  to  45°  C.,  maintained  for  twenty -four  hours. 
But  at  ordinary  temperatures  they  retain  their  toxic  action  for  several 
weeks. 

Gruber  (1892)  has  also  obtained  a  toxic  albuminous  precipitate  by 
allowing  egg  cultures  to  fall  into  alcohol,  drying  the  precipitate,  and 
then  extracting  it  with  water. 

Gamaleia  (1893)  has  obtained  a  toxin  which  produces  the  typical 
phenomena  of  cholera,  which,  according  to  him,  is  closely  associated 
with  the  bacteria  cells,  but  can  be  extracted  by  a  soda  solution  or 
by  heating  to  55°  to  60°  C.  The  conclusion  is  reached  that  it  is  a 
nucleo-alburnm  analogous  to  the  toxalbumins  of  tetanus  and  of 


204  PROTECTIVE   INOCULATIONS. 

diphtheria.  It  is  precipitated  by  alcohol,  acids,  and  by  magnesium 
sulphate. 

Finally,  Westbrook,  in  a  still  more  recent  research  (1894),  arrives 
at  the  conclusion  that  the  cholera  spirillum  produces  various  toxio 
proteids  which  in  small  amounts  produce  immunity  in  susceptible 
animals,  and  the  production  of  which  depends  to  a  certain  extent 
upon  the  culture  medium ;  or  that  its  toxin  is  a  substance  of  constant 
chemical  composition  which  is  mixed  with  various  albuminous  sub- 
stances, either  contained  in  the  culture  medium  or  developed  in  the 
culture.  Duclaux  is  of  the  opinion  that  the  last  supposition  is  cor- 
rect, and  that  the  so-called  toxalbumins  are  not  bodies  of  definite 
chemical  composition,  but  mixtures  of  toxins  and  albuminous  sub- 
stances. 

Experiments  made  upon  the  lower  animals  show  that  the  intro- 
duction of  these  cholera  toxins  into  the  body  of  a  susceptible  animal, 
either  with  or  without  the  living  cholera  spirillum,  results  in  the 
establishing  oi:  a  certain  degree  of  immunity  against  the  toxic  action 
of  cholera  cultures .  And  there  is  good  reason  to  believe  that  a  non- 
fatal  attack  of  cholera  in  man  gives  the  individual  a  relative  immunity 
from  subsequent  attacks,  for  some  time  at  least.  This  has  led  to  ex- 
tended experiments  with  reference  to  the  possibility  of  producing  a 
similar  immunity  in  man  by  means  of  protective  inoculations.  The 
experiments  bearing  upon  this  point  which  have  been  made  upon  the 
lower  animals  will  first  engage  our  attention. 

Hueppe  (1887)  first  demonstrated  the  fact  that  injection  of  a  small 
amount  of  a  cholera  culture  into  the  peritoneal  cavity  of  a'  guinea-pig 
is  fatal  to  these  animals. 

In  the  following  year  (1888)  Gamaleia  reported  his  success  in  in- 
fecting guinea-pigs  by  subcutaneous  injections  of  blood  from  an  in- 
fected pigeon.  He  found  that  by  successive  inoculations  in  pigeons 
a  considerable  increase  in  virulence  is  established;  and  that  while 
guinea-pigs  were  not  fatally  infected  by  subcutaneous  inoculations 
with  ordinary  cultures,  they  invariably  died  when  inoculated  with  the 
more  virulent  culture  in  the  blood  of  an  infected  pigeon.  Also,  that 
when  guinea-pigs  were  inoculated  with  ordinary  cultures,  or  with 
cultures  sterilized  by  heat,  they  were  subsequently  immune,  and  re- 
sisted inoculations  with  the  most  virulent  material.  In  the  same 
year  the  author  referred  to  announced  the  discovery  of  a  spirillum 
which  closely  resembles  the  cholera  spirillum — his  "Yibrio  Metch- 
nikovi."  This  was  obtained  from  the  intestinal  contents  of  fowls 
suffering  from  a  fatal  infectious  malady  (in  Odessa).  According  to 
Gamaleia,  chickens  and  pigeons  which  have  survived  an  inoculation 


PROTECTIVE   INOCULATIONS.  295 

with  a  culture  of  this  spirillum  are  subsequently  immune  against  the 
pathogenic  action  of  the  cholera  spirillum,  and  vice  versa.  In  subse- 
quent communications  Gamaleia  reported  that  sterilized  cultures  of 
his  "Vibrio  Metchnikovi "  (sterilized  by  heat  at  120°  C.)  were  very 
pathogenic  for  rabbits,  fowls,  pigeons,  and  even  for  dogs  and  sheep. 
The  rabbit  proved  to  be  the  most  susceptible  animal,  and  succumbed 
to  doses  of  four  cubic  centimetres  in  from  twelve  to  twenty  hours. 
Doses  of  one  cubic  centimetre  per  one  hundred  grammes  of  body 
weight  caused  a  temporary  indisposition  followed  by  immunity. 
Pigeons  were  made  immune  by  larger  doses. 

The  researches  of  Pfeiffer  (1889)  confirmed  those  of  Gamaleia  as 
to  the  fact  that  pigecns  and  guinea-]  >igs  could  be  made  immune 
against  Vibrio  Metchnikovi  by  the  injection  of  sterilized  cultures. 
But  guinea-pigs  which  had  been  immunized  against  this  pathogenic 
spirillum  succumbed  to  cholera  infection;  and,  on  the  other  hand, 
animals  which  had  been  treated  in  various  ways  with  a  cholera  cul- 
ture died  without  exception  when  infected  with  Vibrio  Metchnikovi. 
The  conclusion  is  therefore  reached  that  the  two  pathogenic  spirilla 
are  distinct  species,  although  very  similar  in  many  respects. 

Brieger  and  \Vassermann  (1892)  have  reported  the  results  of  ex- 
periments with  the  cholera  spirillum  cultivated  in  thymus  bouillon. 
After  twenty -four  hours'  development  in  this  medium  the  cultures 
were  sterilized  by  heat  (55°  C.  for  fifteen  minutes)  and  placed  in  an 
ice-chest  for  twenty-four  hours.  Four  cubic  centimetres  of  this  fluid 
injected  daily  for  four  clays  into  the  peritoneal  cavity  of  a  guinea-pig 
made  it  immune  to  the  cholera  spirillum  in  doses  three  times  as  large 
as  were  required  to  kill  an  animal  not  so  treated.  This  immunity 
lasted  for  two  months.  Fedoroff  (1892)  obtained  similar  results  by 
the  subcutaneous  injection  of  sterilized  cultures  in  doses  of  one  cubic 
centimetre,  in  guinea-pigs.  His  cultures  in  thymus  bouillon  were 
kept  for  from  seven  to  ten  days  at  37°  C.,  then  sterilized  by  heating 
for  fifteen  minutes  at  65°  C.,  then  allowed  to  stand  in  a  dark  room  for 
twenty-four  hours,  and  finally  mixed  with  an  equal  volume  of  glycerin. 

Ketscher  (1892)  has  obtained  evidence  that  the  immunizing  sub- 
stance in  animals  which  have  received  protective  inoculations  is  con- 
tained in  the  milk  of  females  thus  treated.  Three  goats  received 
subcutaneous  inoculations  of  virulent  cholera  cultures,  and  also  injec- 
tions into  a  vein  and  into  the  peritoneal  cavity.  The  milk  of  these 
goats  was  injected  into  the  peritoneal  cavity  of  rabbits ;  these  proved 
to  be  immune  when  subsequently  lethal  doses  of  a  virulent  cholera 
culture  were  injected  into  the  peritoneal  cavity. 

According  to  Gamaleia  (1892),  dogs  are  very  susceptible  to  infec- 


296  PROTECTIVE   INOCULATIONS. 

tion  with  cholera  spirilla,  and  present  symptoms  closely  resembling 
those  of  cholera  in  man.  They  may  also  be  easily  immunized  against 
the  pathogenic  action  of  cholera  cultures. 

Gruber  and  Wiener  (1892)  have  also  found  that  susceptible  ani- 
mals are  easily  immunized  against  cholera  infection  either  by  inocu- 
lation with  small  doses,  with  attenuated  cultures,  or  with  larger  quan- 
tities of  sterilized  cultures.  Haffkine  (1892)  also  reports  his  success 
in  immunizing  guinea-pigs  and  pigeons. 

Pawlowsky  (1893)  claims  to  have  obtained  from  the  blood  of  ani- 
mals having  an  acquired  immunity  against  cholera  an  antitoxin  in  the 
form  of  an  amorphous  powder;  and  Lazarus  (1892)  reports  that  the 
blood  of  man,  after  recovery  from  an  attack  of  cholera,  has  the  prop- 
erty of  protecting  guinea-pigs  from  fatal  infection  when  injected,  in 
very  small  amount,  into  the  peritoneal  cavity.  Issaeff  (1894)  in  an 
extended  series  of  experiments  was  not  able  entirely  to  confirm  the 
results  reported  by  Lazarus.  In  a  summary  of  results  obtained  in 
his  own  experiments  he  says : 

"1.  The  intraperitoneal  or  subcutaneous  injection  of  blood  serum  from 
normal  individuals  [that  is,  persons  who  have  not  suffered  an  attack  of 
cholera],  and  also  of  various  acids,  alkalies,  and  neutral  liquids,  gives  to 
guinea-pigs  a  certain  resistance  against  intraperitoneal  cholera  infection. 
This  resistance,  however,  is  feeble  and  temporary,  aud  cannot  be  considered 
as  identical  with  the  true  immunity  which  results  from  vaccination  with  the 
products  of  the  cholera  bacteria. 

"2.  Guinea-pigs  vaccinated  against  cholera  have  no  immunity  against 
the  toxins  of  the  cholera  vibrio,  notwithstanding  their  high  degree  of  insus- 
ceptibility to  infection  with  cultures  containing  the  living  vibrio.  The  blood 
of  immunized  guinea-pigs  does  not  possess  antitoxic  properties.  The  maxi- 
mum dose  of  cholera  toxins  which  immune  guinea-pigs  can  withstand  is  not 
greater  than  that  which  control  animals  withstand. 

"3.  The  blood  of  guinea-pigs  carefully  immunized  against  cholera  pos- 
sesses specific  and  very  pronounced  immunizing,  and,  in  a  certain  sense, 
curative  powers. 

"4.  The  blood  of  cholera  convalescents  possesses  similar  specific  and 
curative  powers.  This  property  is  first  developed  about  the  end  of  the  third 
week  after  the  attack,  and  disappears  completely  at  the  end  of  two  or  three 
months." 

In  a  series  of  experiments  made  by  Pfeiffer  and  Issaeff  the  results 
obtained,  as  stated  by  Pfeiffer  in  a  subsequent  communication,  were 
as  follows : 

"In  my  research  with  Issaeff  'upon  the  explanation  of  cholera  immu- 
nity '  I  proved  that  the  serum  of  animals  which  have  an  active  acquired  im- 
munity against  cholera  only  has  a  specific  action  upon  this  particular  species 
of  vibrio,  and  as  regards  other  species  of  bacteria  does  not  differ  in  its  action 
from  the  blood  serum  of  normal  animals.  We  also  showed  that  this  specific 
influence  in  respect  to  the  intraperitoneal  cholera  infection  of  guinea-pigs 
was  due  exclusively  to  bactericidal  processes  which  in  some  way  were  in- 
duced by  the  serum  of  immune  animals." 


PROTECTIVE   INOCULATIONS.  297 

The  view  of  Pfeiffer,  founded  upon  his  experimental  results,  is 
that  the  destruction  of  the  living  cholera  spirilla,  which  quickly  takes 
place  in  the  peritoneal  cavity  of  the  guinea-pig,  when  at  the  same 
time  a  minute  quantity  of  serum  from  an  immune  animal  is  intro- 
duced, is  not  directly  due  to  the  bactericidal  action  of  this  serum,  but 
that  in  some  way  it  gives  rise  to  a  specific  bactericidal  action  in  the 
exudate  which  is  found  in  the  peritoneal  cavity  as  a  result  of  such  in- 
jections. His  experiments  also  lead  him  to  the  conclusion  that  this 
is  accomplished  quite  independently  of  phagocytosis. 

The  brief  review  of  experimental  researches  relating  to  cholera 
immunity  which  we  have  made  shows  that,  while  there  is  a  general 
agreement  as  to  the  possibility  of  producing  immunity  in  susceptible 
animals,  there  is  considerable  difference  of  opinion  as  to  the  true  ex- 
planation of  this  immunity.  The  supposition  that  it  is  due  to  an 
antitoxin  which  has  the  power  of  neutralizing  the  toxic  products  of 
the  cholera  spirillum  does  not  receive  any  support  from  the  most  re- 
cent investigations — those  of  Pfeiffer  and  Issaeff — which,  on  the  con- 
trary, seem  to  establish  the  fact  that  this  immunity  depends  upon  an 
increased  bactericidal  activity  of  the  blood  serum  of  immune  animals. 
A  very  curious  fact  developed  by  the  researches  of  the  bacteriologists 
last  named  is  that — 

"The  cholera  serum  which  in  the  peritoneal  cavity  of  guinea-pigs  acted 
only  upon  the  cholera  bacteria,  and  behaved  toward  other  vibrios  exactly  like 
the  serum  of  normal  animals,  in  a  test  tube  killed  all  four  species  of  vibrios 
with  equal  rapidity." 

Unfortunately  the  evidence  relating  to  the  value  of  protective  in- 
oculations in  man,  although  supported  by  the  evidence  already  re- 
ferred to  as  regards  the  lower  animals,  is,  to  a  considerable  extent, 
unsatisfactory,  owing  to  the  difficulty  of  applying  scientific  methods 
to  experiments  of  this  kind.  The  evidence,  however,  is  in  favor  of 
the  view  that  a  certain  degree  of  protection  is  afforded  by  the  subcu- 
taneous injection  of  cholera  cultures.  Such  protective  inoculations 
could  not  be  expected  to  confer  an  absolute  immunity,  inasmuch  as 
the  immunity  resulting  from  a  single  attack  has  only  a  relative  value, 
and  is  probably  not  of  long  duration. 

We  quote  from  Shakespeare's  "Eeport  on  Cholera  in  Europe  and 
India,  1890,"  the  following  paragraphs  relating  to  immunity  as  a 
result  of  an  attack  of  cholera : 

"IMMUNITY  AFTER  AN  ATTACK  OF  CHOLERA — EXPERIENCES  IN 

FRANCE,    1884. 

"The  Academy  of  Medicine  of  Paris  directed  a  circular  letter  of  questions 
concerning  cholera  to  the  physicians  of  the  localities  infected  by  that  disease 


298  PROTECTIVE   INOCULATIONS. 

iii  1884,  and  in  group  L  of  general  observations  in  that  questonario  is  found 
the  following :  '  Have  there  been  observed  recurrences  among  the  people 
attacked,  either  in  a  former  epidemic  or  in  the  present  one  ?  Give  the  results 
of  this  recurrence.'  In  response  to  their  questions  the  Academy  received  184 
communications,  but  the  committee  appointed  to  analyze  them  eliminated 
79  ;  for  various  .reasons  given  only  104  were  used  for  analysis.  Of  this  num- 
ber only  8  bore  upon  the  particular  question  above  mentioned,  and  it  is 
reasonable  to  assume  that  the  other  96  observers  said  nothing  concerning  this 
point  because  they  had  observed  nothing  bearing  upon  it.  The  results  of 
this  analysis  may  be  stated  as  follows  : 

"From  Castelnaudary,  with  a  population  of  10,000,  we  learn  that  there 
were  54  cases  and  18  deaths  from  cholera,  among  which  there  was  1  recur- 
rence ;  from  Aix,  with  20,257,  number  of  cases  unknown,  deaths,  117,  among 
these  2  recurrences  were  observed,  at  intervals  of  ten  and  forty  days  ;  from 
Beseges,  with  11,400  inhabitants,  we  learn  of  124  cases  and  40  deaths,  among 
which  were  2  recurrences  ;  from  Cette,  with  35,000,  the  number  of  cases  is 
not  mentioned,  but  we  learn  that  there  were  92  deaths  and  1  recurrence ; 
from  Nantes,  with  124,300  inhabitants  we  learn  of  251  cases  and  112  deaths, 
with  1  recurrence  ;  from  Perpignaii,  with  25,000  inhabitants,  we  hear  of  325 
cases  and  225  deaths,  and  receive  the  indefinite  statement  that  there  were 
some  fatal  recurrences  ;  from  Pigiiaiis,  population  not  stated,  we  learn  of  22 
attacks  and  12  deaths,  with  1  recurrence  ;  from  Cadenet,  with  a  population 
of  26,000,  we  are  not  informed  of  the  number  of  cases,  but  learned  that  there 
were  20  deaths  and  2  recurrences." 

"IMMUNITY  AFTER  AN  ATTACK  OF  CHOLERA — EXPERIENCE  IN 

SPAIN,  1885. 

"  While  examining  cholera  in  Spain,  the  writer  prepared  a  circular  con- 
taining a  series  of  twenty-five  questions  relating  especially  to  the  nature,  eti- 
ology, and  prophylaxis  of  cholera,  one  of  which  requested  the  physician  to 
state  whether  or  not,  in  his  own  personal  experience,  he  had  observed  a  sec- 
ond or  a  third  attack  of  cholera  during  the  same  epidemic,  and  in  case  of  a 
positive  reply  to  detail  the  symptoms  and  all  the  circumstances  surrounding 
it.  This  circular-letter  was  addressed  to  some  twenty-five  hundred  Spanish 
physicians,  located  in  the  various  cities,  towns,  and  villages  in  that  kingdom 
which  had  suffered  from  the  epidemic.  Among  the  large  number  of  replies 
there  were  only  eight  in  which  a  second  attack  was  reported,  and  from  an  ex- 
amination of  the  details  of  these  there  was  110  doubt  left  in  our  mind  that  six 
were  not  genuine  second  attacks  after  a  complete  recovery,  but  were  in 
reality  relapses  due  to  imprudences  of  diet  or  otherwise  before  convalescence 
and  complete  recovery  had  been  established.  Two  of  the  eight  cases,  from  the 
details  of  the  reports  given,  may  have  been  genuine  recurrent  attacks  of 
Asiatic  cholera,  or  may  have  been  simply  seizures  of  cholera  morbus  (cholera 
nostras).  It  is  well  knowrii  that  after  an  attack  of  Asiatic  cholera  the  di- 
gestive apparatus  is  left  in  a  damaged  condition,  and  disorders  of  the  in- 
testines continue  for  a  long  time.  The  habits  of  life  and  the  imprudences  so 
common  to  the  class  of  people  most  frequently  suffering  from  Asiatic  cholera 
in  that  country  are  such  as  to  render  them  more  than  usually  liable  to  suffer 
attacks  of  cholera  nostras.  As  having  an  important  bearing  upon  this  sug- 
gestion, the  writer  made  an  analysis  of  the  vital  statistics  of  Spain,  covering 
the  five  years  previous  to  1885,  for  the  purpose  of  learning  the  extent  of 
prevalence  of  cholera  nostras  among  that  population,  and  the  result  of  the  in- 
quiry shows  that  the  number  of  deaths  attributed  to  that  disease  averaged 
per  year  sixteen  per  every  million  inhabitants." 

Dr.  Ferran,  who  practised  inoculations  on  an  extensive  scale  dur- 
ing the  epidemic  of  1885,  in  Spain,  gives  the  following  account  of  his 
method  of  performing  these  inoculations : 


PROTECTIVE   INOCULATIONS.  299 

"  1.  The  cholera  vaccine  is  nothing  more  than  a  pure  culture,  in  bouillon, 
of  the  comma  bacillus.  Its  easy  and  long  preservation  (four  to  five  days) 
allows  of  its  transportability  to  great  distances,  taking  care  always  to  keep 
the  flask  which  contains  the  material  upright. 

"  2.  Heat  and  cold  do  not  interfere  with  its  preservation  if  the  vaccine  is 
to  be  used  in  a  short  time.  It  should  not,  however,  be  kept  out  of  doors  dur- 
ing the  warm  season. 

"3.  The  vaccine  should  be  kept  in  flasks  of  the  model  of  Ferraii,  with  a 
flat  bottom  and  a  short  neck.  The  stopper,  which  is  of  rubber,  fits  perfectly, 
and  is  penetrated  by  two  glass  tubes.  One,  straight  and  short,  which  does  not 
extend  below  the  inferior  surface  of  the  stopper,  and  which  does  not  project 
above  more  than  some  two  centimetres,  is  plugged  with  a  small  quantity  of 
sterilized  cotton  and  a  superficial  covering  of  wax.  The  other  glass  tube  is 
longer,  and  extends  on  the  lower  side  as  far  as  the  bottom  of  the  flask,  while 
its  superior  end  is  curved,  and  terminates  in  a  capillary  extremit3r,  the  tip  of 
which  is  closed  with  wax. 

"4.  When  the  vaccine  is  to  be  used  it  is  necessary  to  make  two  principal 
preparations  for  the  operation.  A  small  syringe  for  the  hypodermic  injection, 
and  a  small  vessel  into  which  it  is  necessary  to  empty  the  fluid  from  the  flask, 
are  required.  The  syringe  should  have  metallic  pistons  and  mountings, 
without  mastic  of  any  kind  and  without  rubber.  Its  capacity  should  be  one 
cubic  centimetre,  its  needle  thicker  and  shorter  than  that  of  ordinary  use. 
Before  beginning  the  vaccination  the  syringe  must  be  filled  two  or  three 
times  with  boiling  water,  which  is  aspirated  and  expelled  through  the  needle. 
This  is  called  sterilizing  the  instrument,  and  by  this  means  the  extraneous 
-••mis  are  destroyed  which  might  be  contained  in  it,  in  order  to  avoid  the 
production  of  phlegmons  and  abscesses.  The  trouble  in  taking  this  precaution 
will  be  little.  Acting  thus,  one  may  perform  thousands  of  injections  without 
fear  of  any  accident.  It  is  suggested  that  it  is  a  bad  custom  to  pass  the  nee- 
dle through  a  flame  in  order  to  sterilize  it,  because  this  mode  of  procedure 
draws  the  temper.  Another  precaution  that  must  be  taken  relates  to  the 
examination  of  the  syringe  before  using  it,  in  order  to  be  well  assured  that  the 
piston  acts  perfectly  and  that  not  a  single  drop  of  the  liquid  escapes  by  a  leak 
in  the  cannula.  This  latter  defect  is  sufficient  to  reject  the  instrument.  If  the 
syringe  aspires  air  because  the  leather  washer,  which  is  placed  at  the  end  of 
the  glass  tube  in  order  to  facilitate  its  adaptation,  is  dry,  or  the  piston  is  in  the 
same  condition,  it  is  necessary  to  delay  a  little  while  in  order  to  take  the 
syringe  apart  and  soak  it  in  warm  water.  It  is  convenient  to  keep  several 
syringes  for  use,  with  a  sufficient  number  of  needles,  when  many  inocula- 
tions are  to  be  performed. 

"5.  The  small  receptacle  into  which  the  vaccine  is  poured  in  order  that 
the  syringe  may  be  filled  readily  is  a  capsule,  a  cup,  or  some  similar  vessel. 
Before  use,  it  should  be  washed  and  dried  with  extreme  care,  and  imme- 
diately before  using  passed  through  an  alcohol  or  Bunsen  flame,  in  order  to 
sterilize  it. 

"6.  All  these  preparations  having  been  made,  the  drop  of  wax  which 
closes  the  capillary  extremity  of  the  long  tube  of  the  flask  is  removed,  and  at 
the  same  time  also  the  wax  covering  of  the  cotton  stopper  of  the  short  tube, 
but  by  no  means  must  this  cotton  stopper  be  removed  ;  a  rubber  tube,  or  the 
extremity  of  a  small  Richardson  spray  apparatus,  is  adjusted  to  the  short 
tube.  The  capillary  extremity  of  the  long  tube  is  now  slightly  warmed  in 
order  to  soften  somewhat  the  wax  which  may  have  been  drawn  into  its 
lumen  by  capillarity,  and  air  is  forced  into  the  flask,  either  by  blowing  into 
the  rubber  tube  or  by  working  the  Richardson  atomizer  ;  the  air  injected  by 
pressure  upon  the  vaccine  fluid  forces  the  latter  out  through  the  long  tube 
with  the  capillary  extremity,  and  it  is  collected  in  the  cup  or  small  sterilized 
vessel.  This  latter  is  then  covered  with  white  paper,  which  has  been 
scorched  in  the  flame,  or  with  a  sterilized  glass  plate  ;  as  often  as  the  syringe 
is  filled  this  cover  will  be  removed  and  again  immediately  afterward  replaced. 


300  PROTECTIVE   INOCULATIONS. 

"7.  Never  should  the  rubber  stopper  which  closes  the  flask,  or  the  cotton 
which  plugs  the  short  straight  tube,  be  removed,  because  otherwise  the 
germs  of  the  external  air  might  enter  and  contaminate  the  culture,  and  in 
this  way  give  place  to  local  and  general  accidents  among  the  inoculated. 
Whenever,  through  the  movements  of  transportation,  the  cotton  plug  in  the 
short  glass  tube  has  become  so  wet  as  to  impede  the  passage  of  the  air  which 
is  to  be  forced  into  the  flask  in  the  act  of  expelling  the  vaccine  from  it,  it 
may  be  removed  writh  the  point  of  a  needle  and  rapidly  substituted  by  an- 
other plug  of  surgical  cotton  which  has  been  carbonized  or  salicylized.  If 
this  proceeds  with  cleanness  and  promptness,  there  is  no  danger  in  doing  it. 
When  the  cotton,  although  wet,  does  not  impede  the  injection  of  the  air,  it  is 
better  not  to  change  it. 

"8.  After  terminating  the  vaccination,  again  the  capillary  extremity  of 
the  curved  tube  is  passed  through  the  flame  until  the  small  quantity  of  liquid 
remaining  in  it  is  evaporated  ;  it  is  then  stopped  a  second  time  with  a  small 
drop  of  wax  ;  and  from  the  other  glass  tube  the  rubber  tube  which  has  been 
employed  for  forcing  in  the  air  is  removed  and  another  thin  layer  of  wax  is 
placed  over  the  cotton  plug. 

"9.  If  in  the  smaller  vessel  or  cup  any  of  the  vaccine  fluid  remains  after 
the  vaccination  of  all  persons  present,  it  is  boiled,  and  in  this  manner  the 
culture  is  killed,  for  it  should  not  be  used  in  another  operation,  because  at- 
mospheric germs  might  become  mixed  with  it. 

' '  10.  The  technique  for  the  practice  of  the  inoculation  is  the  same  as  for  all 
hypodermic  injections.  The  most  convenient  region  is  that  of  the  brachial 
triceps. 

"  11.  The  dose  is  one  cubic  centimetre — or  the  contents  of  a  syringe — into 
each  arm,  for  individuals  of  all  ages  and  conditions.  / 

"  12.  Five  days  having  elapsed,  re  vaccinations  may  be  performed  by  fol- 
lowing the  same  instructions." 

Shakespeare,  who  was  sent  by  the  United  States  Government  to 
Spain  to  investigate  the  results  of  these  inoculations,  reports  as  fol- 
lows: 

"  And  now  with  respect  to  the  human  inoculations:  The  most  of  these 
inoculations  were  performed  in  villages  in  the  province  of  Valencia.  The 
number  of  persons  inoculated  considerably  exceeds  thirty  thousand.  Much 
has  been  both  said  and  written  in  Spain,  France,  and  England  concerning 
the  results  of  these  inoculations.  The  results  which  have  been  published 
have  appeared  to  very  strongly  back  up  the  claim  of  Dr.  Ferrari  that  chol- 
eraic inoculation  has  the  power  of  protecting  the  individual  against  an  at- 
tack of  cholera,  and  that  the  extensive  practice  of  this  inoculation  among 
villages  already  invaded  by  the  epidemic  is  a  powerful  and  at  the  same  time 
harmless  means  of  bringing  the  epidemic  to  an  end.  This  being  the  case, 
for  those  who  were  unwilling  to  accept  the  deductions  to  be  made  from  the 
published  statistics  the  only  way  of  escaping  their  force  seemed  to  be  by 
an  attack  upon  their  validity. 

"The  statistics  of  the  anti-choleraic  inoculations  have  been  widely  at- 
tacked. The  first  public  onslaught  upon  these  statistics  of  which  the  world, 
outside  of  Spain,  had  much  knowledge  was  made  in  the  report  of  the  French 
Commission,  with  Dr.  Brouardel  at  its  head,  which  was  presented  to  the 
Minister  of  Commerce  after  the  return  of  that  Commission  from  Spain  in  the 
summer  of  1885.  It  is  charged  in  that  report  that  the  results  of  the  statistics 
therein  reproduced  are  assailable  on  account  of  having  been  collected  by 
physicians  who  were  partisan  supporters  of  Dr.  Ferran,  and  that  they  neither 
possessed  any  adequate  official  character  nor  did  they  possess  sufficient  de- 
tails. As  far  as  I  can  learn,  the  general  impression  entertained  throughout 
the  world  of  the  value  of  inoculation  statistics  is  based,  in  the  main,  upon 
this  report  of  the  French  Commission. 


PROTECTIVE   INOCULATIONS.  301 

"The  statement  of  that  Commission  that  the  statistics  which  they  had 
been  able  to  obtain  of  the  preventive  inoculations  of  Ferrari  were  to  a  con- 
siderable degree  void  of  any  official  character  may  be  true,  and  perhaps  it  is 
also  true  that  they  emanated  from  the  partisan  friends  of  Ferra.ii ;  but  it  must 
be  distinctly  remembered  that  at  that  day  there  were  practically  no  official 
statistics  of  this  kind  in  the  hands  of  any  one.  The  official  statistics  collected 
under  the  orders  of  the  Spanish  Government  were  gotten  together  at  a  far 
later  date. 

' '  Upon  the  appointment  of  the  Government  at  Madrid  of  the  second  offi- 
cial Spanish  Commission  to  investigate  the  Ferran  question  in  the  provinces 
where  the  inoculations  were  being  practised,  it  was  ordered  that  official  sta- 
tistics of  inoculation  should  be  collected  in  the  usual  manner  ;  that  is  to 
say,  by  the  customary  statistical  officers  of  the  Government.  This  second 
medical  Commission  was  also  accompanied  by  an  independent  statistical 
commission  who  were  charged  with  the  duty  of  forming  statistics  of  those 
inoculations  which  were  expected  to  be  witnessed  by  the  Medical  Commission 
in  their  tour  of  investigation,  and  the  report  to  the  Spanish  Government  of 
this  statistical  commission  is  based  exclusively  upon  the  official  statistics 
which  they  themselves  collected. 

' '  In  estimating  the  value  of  the  official  character  and  the  authority  of  the 
official  statistics,  which  have  since  the  visit  of  the  French  Commission  to 
Spain  been  collected  and  published,  the  following  circumstances  should  be 
taken  into  account :  The  provincial  governments  of  Spain  are  somewhat 
peculiar,  in  that  the  civil  governors  change  with  the  changes  which  take 
place  in  the  Government  at  Madrid,  so  that  the  political  constitution  of  the 
provincial  governments  is  always  a  reflex  of  that  of  the  central  government 
at  Madrid.  Moreover,  the  political  sentiment  of  the  provincial  government 
is  also  more  or  less  perfectly  reflected  by  the  local  governments  of  the  towns 
of  the  province. 

"  The  hostility  of  the  Minister  of  the  Interior  at  Madrid  to  Dr.  Ferr'dn,  and 
his  attempts  at  the  prevention  of  cholera  by  inoculation,  is  a  well-known  fact 
now  generally  admitted  ;  and  the  hostility  which  Dr.  Ferran  met  with  from 
the  civil  governor  of  the  province  of  Valencia  was  even  greater  than  that 
manifested  by  the  Minister  of  the  Interior  himself. 

"The  official  statistics  of  the  Ferrari  inoculations  are  in  the  first  place 
signed  by  the  physicians  of  the  locality ;  and  in  the  next  place  by  the  judge 
of  the  municipal  court,  and  sometimes  also  by  the  president  judge  of  the 
judicial  district,  by  the  parochial  priest,  and  by  the  mayor  of  the  municipality, 
whose  signatures  and  seals  are  attested  by  an  authorized  notary  public. 

"It  must,  therefore,  be  obvious  that  the  charge  made  by  the  French  Com- 
mission, which  has  been  so  constantly  reiterated  everywhere,  that  the  public 
statistics  of  the  anti-choleraic  inoculations  are  void  of  official  character  and 
are  to  be  regarded  as  ex-parte  testimony  of  the  partisans  of  Ferran,  cannot 
apply  to  official  statistics  which  were  collected  under  the  supervision  of  the 
municipal  authorities  of  the  villages  wherein  the  inoculations  were  per- 
formed, and  attested  not  only  by  the  local  judicial  officers  and  the  parochial 
priests,  but  also  by  the  political  officers — that  is  to  say,  the  secretaries  and 
the  mayors  of  the  municipalities  ;  for  it  must  be  admitted  that  neither  the  po- 
litical officers  of  the  municipalities  nor  of  the  provincial  governments,  any 
more  than  the  parochial  priest,  can  reasonably  be  charged  with  being  the 
partisans  or  friends  of  Ferran — the  Minister  of  the  Interior  continuing  dur- 
ing the  time  of  collection  of  these  official  statistics  to  be  hostile  to  the  claims 
of  Ferran.  It  therefore  follows  that  the  attack  upon  the  statistics  of  the  in- 
oculations made  by  the  French  Commission,  and  so  widely  accepted  by  the 
medical  world  as  conclusive,  does  not  apply  to  the  official  statistics  of  which 
we  are  speaking.  And,  in  view  of  this  fact,  the  evidence  as  to  the  efficiency 
and  harmlessness  of  the  anti-choleraic  inoculations  should  be  re-examined. 
As  I  have  already  said,  the  results  of  the  preventive  inoculations  of  Ferran 
as  set  forth  in  the  official  statistics  appear  to  very  strongly  support  his  claim 


302  PROTECTIVE   INOCULATIONS. 

of  the  protective  value  of  the  inoculations.  Iii  view  of  the  great  importance 
of  this  whole  subject,  I  have  determined  to  place  these  statistics  in  this  report 
for  the  benefit  of  the  readers  of  the  English  language,  in  order  that  they  mav 
judge  for  themselves  of  the  facts  as  they  appear  to  be  recorded. 

"From  the  Government  statistics  of  cholera  throughout  the  province  of 
Valencia,  it  appears  that  among  the  villages  invaded  there  were  62  attacks 
per  one  thousand  of  the  population,  and  31  deaths  per  thousand,  which  gives 
a  mortality  of  50  per  cent  of  those  attacked.  It  appears  from  analysis  of 
the  published  official  statistics  of  cholera  in  22  towns  where  inocula- 
tion was  performed  the  inhabitants  were  divided  as  follows:  104,561  not 
inoculated  ;  30,491  inoculated.  Of  the  latter  there  were  387  attacks  of  cholera, 
or  12  per  thousand,  and  104  deaths,  or  3  per  thousand  ;  the  mortality  of  those 
attacked  being  25  per  cent.  Of  the  former  there  were  8,406  attacks, 
or  77  per  thousand,  and  3,512  deaths,  or  33  per  thousand,  being  a  mortality 
of  those  attacked  of  43  per  cent.  It  appears,  therefore,  that  among 
the  population  of  villages  wherein  anti-choleraic  inoculations  had  been  more 
or  less  extensively  performed  the  liability  of  the  inoculated  to  attacks  of 
cholera  was  6.06  times  less  than  that  of  the  non-inoculated,  whilst  the  liability 
of  the  inoculated  to  death  by  cholera  was  9.87  times  less  than  that  of  the  11011- 
inoculated.  These  figures  are  based  exclusively  upon  the  data  furnished  by 
inoculations,  the  reiiioculatioiis  being  left  out  of  consideration,  because  they 
are  much  less  numerous,  although  from  the  records  of  the  inoculations  it 
would  seem  that  the  liability  of  attack,  and  especially  of  death  by  cholera,  is 
many  times  less  among  them  than  among  those  inoculated  a  single  time. 

•'The  charge  has  also  been  made  with  respect  to  the  published  records  of 
the  inoculations  that  the  hygienic  and  physical  condition  of  the  subjects  of 
inoculation  have  not  been  sufficiently  indicated  in  the  records,  and  that  the 
vast  majority  of  those  profiting  by  the  opportunity  to  receive  the  aiiti- 
choleraic  inoculations  were  of  the  middle  and  upper  classes,  and  therefore 
not  of  that  class  of  inhabitants  who  are  notoriously  most  liable  to  attack  and 
death  from  cholera.  This  criticism  may  have  some  justness  as  respects  some, 
perhaps  many,  of  the  villages  where  inoculations  were  performed  ;  but  there 
are  certainly  many  of  the  villages  wherein  the  results  of  the  inoculation 
seemed  to  be  most  positively  in  favor  of  the  claim  of  Ferran  where  this 
criticism  cannot  hold.  I  refer  to  villages  wherein  three-fourths  or  four- 
fifths  of  the  inhabitants  were  inoculated,  leaving  only  the  fraction  of  the 
population  non-inoculated.  Even  in  the  absence  of  any  special  notes  indi- 
cating the  social  conditions  and  hygienic  surroundings  of  the  inoculated  in 
these  villages,  it  is  ridiculous  to  assume  that  the  vast  majority  of  these  were 
people  of  the  middle  and  upper  classes,  and  were  therefore  but  little  liable  to 
attack  and  death  by  cholera.  Any  one  acquainted  with  the  character  of  the 
Spanish  population  as  it  exists  in  the  rural  villages  will  admit  at  once  that 
the  vast  majority  of  this  population  consists  of  the  wretched  and  the  poor, 
who  live  under  the  most  unhygienic  and  unsalubrious  conditions,  and  there- 
fore are  of  that  class  most  liable  to  suffer  from  cholera. 

"There  is  still  another  result  of  the  preventive  inoculations  of  Ferran 
apparently  shown  by  these  statistics.  I  refer  to  the  apparent  marked  short- 
ening of  the  course  of  the  epidemic  after  a  large  percentage  of  the  inhabitants 
had  become  inoculated.  It  would  seem,  therefore,  from  analysis  of  the 
official  statistics,  that  the  practice  of  the  an ti -choleraic  inoculation  after  the 
method  of  Ferran,  besides  giving  the  subject  inoculated  a  considerable  im- 
munity from  attack  and  death  by  cholera,  furnishes  a  means  of  bringing  an 
epidemic  rapidly  to  an  end." 

With  reference  to  Haffkine's  method  of  inoculation  we  cannot  do 
better  than  to  quote  from  a  lecture  which  lie  gave  in  London,  in  1893 : 

"In  the  research  that  I  have  done  at  the  Pasteur  Institute  on  vaccination 
against  Asiatic  cholera  I  have  chosen  for  my  starting-point  the  inoculation 


PROTECTIVE   INOCULATIONS.  303 

of  the  animal  into  the  peritoneal  cavity.  Starting  from  this  point  I  have 
worked  out  a  method  which  permits  the  culture  of  the  microbe  in  the  animal 
organism  in  a  state  of  purity  during  indefinite  generations,  the  exaltation  of 
it  to  a  well-determined  maximum  of  strength,  and  keeping  it  at  the  same 
degree  of  virulence  for  an  unlimited  period  of  time. 

"This  method  is  illustrated  by  three  series  of  experiments  which  were  the 
subject  of  our  publications  in  the  Comptes  rendus  de  la  Societ£  de  Biologie 
of  Paris,  and  which  are : 

"  1.  Giving  the  first  animal  a  dose  larger  than  the  fatal  dose,  and  killing 
this  animal  in  a  sufficiently  short  space  of  time  to  be  able  to  find  the  more 
resisting  microbes. 

"2.  To  expose  the  exudation  taken  from  the  peritoneal  cavity  to  the  air 
for  several  hours. 

"3.  Then  to  transfer  this  exudation  to  the  next  animal,  of  large  or  small 
size,  according  to  the  concentration  of  the  exudation. 

' '  In  the  hands  of  a  number  of  other  experimenters  this  method  has  given 
the  same  results  and  showed  a  perlect  consistency. 

"The  properties  of  the  virus  which  is  obtained  in  this  manner  of  cultiva- 
tion are  as  follows :  Upon  intraperitoneal  inoculation  it  kills  guinea-pigs 
regularly  in  the  space  of  about  eight  hours,  and  the  fatal  dose  for  this  animal 
is  reduced  to  about  twenty  times  less  than  that  which  it  would  have  been 
necessary  to  take  for  the  microbe  with  which  I  started.  The  same  inocula- 
tion kills  rabbits  and  pigeons  with  a  dose  which  would  have  been  perfectly 
harmless  at  the  beginning  of  the  experiments.  It  kills  guinea-pigs  by  intra- 
muscular inoculation. 

"The  subcutaneous  inoculation  brings  about  the  formation  of  a  large 
oedema,  which  tends  toward  sequestration  of  a  whole  part  of  the  cutaneous 
tissues  and  to  the  formation  of  a  wide  open  wound,  which  is  cured  in  from 
two  to  three  weeks. 

"The  basis  of  anticholeraic  vaccination  is  founded  on  the  virus  obtained 
in  the  manner  we  have  just  described. 

"This  virus,  injected  under  the  skin  of  a  healthy  animal,  gives  it,  after 
several  days,  immunity  from  all  choleraic  contamination,  in  whatever  man- 
ner this  may  arise ;  that  is  to  say,  if  an  animal  that  has  been  thus  treated  be 
taken,  and  an  attempt  made  to  infect  it  either  by  the  digestive  canal,  by 
neutralization  of  the  gastric  juice  and  the  injection  of  opium  into  the  peri- 
toneum, or  by  the  introduction  of  the  microbe  into  the  intestines  by  the 
method  of  Nicati  and  Rietsch,  or  by  intramuscular  inoculation,  or  finally, 
by  intraperitoneal  injection,  the  most  terrible  of  all,  it  resists,  whilst  the  con- 
trol animals  succumb. 

"Anticholeraic  vaccination  of  animals  in  this  manner  is  then  definitely 
established.  But  the  operation  described  cannot  be,  such  as  it  is,  applied  to 
man.  The  wound  following  on  the  subcutaneous  inoculation  is  terrible  to 
look  at,  and,  in  all  probability,  extremely  painful.  Besides,  although  it 
does  not  in  itself  present  any  danger  to  the  health  of  the  individual,  it  exposes 
him  to  all  the  complications  inseparable  from  an  open  wound. 

"This  power  of  producing  necrosis  of  the  cutaneous  tissues  has  been 
removed  from  the  exalted  vaccine  by  cultivating  it  at  a  temperature  of  39° 
C.,  and  in  an  atmosphere  constantly  aerated.  Under  these  conditions  the 
first  generations  of  the  cholera  microbe  would  die  rapidly,  in  an  interval  of 
two  to  three  days,  and  therefore  care  must  be  taken  to  sow  them  again  in 
new  media  immediately  before  death,  and  after  a  series  of  generations  of  this 
kind  a  culture  is  obtained  which,  if  injected  under  the  skin  of  animals,  even 
in  exaggerated  doses,  produces  only  a  passing  oedema,  and  prepares  the 
organism  in  such  a  manner  that  the  injection  of  exalted  virus,  the  definite 
vaccine,  only  produces  a  local  reaction  of  the  slightest  description. 


304  PROTECTIVE   INOCULATIONS. 


"VACCINATION  BY  FIXED  VACCINE. 

"The  method  of  vaccination  thus  worked  out  comprises,  then,  two  vac- 
cines— a  mild  vaccine,  obtained  by  weakening"  the  fixed  virus ;  and  a 
strengthened  vaccine,  which  is  presented  by  the  virus  itself.  It  is  easy  to 
understand  why,  to  obtain  the  weakened  vaccine,  we  do  not  use  an  ordinary 
virus,  but  a  virus  the  nature  of  which  has  been  previously  fixed  in  the  labor- 
atory. It  is  because  the  virus,  such  as  is  found  in  the  natural  state,  especially 
when  it  has  a  saprophytic  phase  of  development,  presents  such  pathogenic 
differences  that  there  is  no  certainty  in  its  application.  Respecting  this  we 
need  only  recall  the  story  of  variolization,  and  the  great  danger  that  an  indi- 
vidual incurred  when  the  infectious  substance  from  a  slightly  attacked  sub- 
ject was  transferred  to  him.  The  mildness  or  the  gravity  of  an  infection  does 
not  depend  only  on  the  veritable  strength  of  the  contagious  substance,  but 
upon  the  resistance  of  the  individual  from  whom  it  is  taken.  Thus  it  hap- 
pened that  in  taking  vaccine  lymph  from  a  subject  lightly  affected,  a  very 
weak  substance  was  sometimes  produced,  which  was  incapable  of  producing 
a  protective  action  ;  and  sometimes  a  lymph  of  such  strength  that  it  killed 
less  resistant  individuals.  The  great  benefit  of  Jenhers  discovery  lay  in 
that  it  precisely  indicated  a  substance  fixed  by  passages  through  animals,  and 
of  a  virulence  below  that  which  is  fatal  to  the  human  organism.  Another 
example  is  given  in  the  method  of  Toussaint  of  vaccination  against  anthrax, 
the  first  of  its  kind,  which  has  been  obliged  to  make  way  for  the  method  of 
M.  Pasteur,  for  the  sole  reason  that  the  latter,  based  upon  virus  of  a  fixed 
nature,  presented  an  absolute  certainty  in  its  results  which  was  wanting  in 
the  other.  Finally,  in  the  history  of  cholera  itself  I  may  recall  the  attempt 
made  in  1885  by  Dr.  Ferraii,  of  Barcelona,  who,  with  the  object  of  preserv- 
ing the  population  of  the  Peninsula  from  the  epidemic  of  cholera,  made 
injections  in  his  patients  of  the  ordinary  virus  taken  from  dead  bodies  and 
cultivated  in  the  laboratory.  The  statistics  of  the  results  obtained  by  this 
means  showed  such  uncertainty  that  110  one  dared  to  recommend  this  opera- 
tion to  his  country  in  spite  of  the  very  numerous  trials  made  in  Spain. 

44  The  possibility  of  treating  the  animal  organism  by  vaccines  of  an  abso- 
lutely fixed  nature,  prepared  by  means  of  special  operations,  constitutes,  on 
the  contrary,  the  basis  of  the  Pasteurian  method,  and  here  lies  the  whole 
secret  and  the  sole  guarantee  of  the  success  of  its  application. 

"APPLICATION  OF  THE  METHOD  TO  MAN. 

"The  method  of  anticholeraic  vaccination,  worked  out  by  experiments 
on  guinea-pigs,  was  tried  upon  rabbits  and  pigeons  before  it  was  applied  to 
man.  These  animals  were  chosen  in  order  to  have  subjects  very  differently 
organized,  and  in  order  to  be  able  to  generalize  the  conclusions,  and  to  be 
able  to  extend  them  to  the  human  organism. 

"  The  result  obtained  011  all  these  animals  being  absolutely  the  same,  it 
was  decided  to  apply  the  operation  to  man. 

' '  The  symptoms  produced  by  this  operation  have  been  described  in  several 
scientific  magazines.  The  method  has  been  tried  at  Paris,  Cherbourg,  and 
at  Moscow,  on  about  fifty  persons  of  both  sexes,  between  the  ages  of  nineteen 
and  sixty-eight,  of  French,  Swiss,  Russian,  English,  and  American  nation- 
ality. 

' '  In  every  case  the  method  has  shown  itself  absolutely  harmless  to  health, 
and  the  symptoms  that  it  evoked  were  a  rise  in  temperature,  a  local  sensitive- 
ness at  the  place  of  inoculation,  and  the  formation  of  a  transitory  oedema  at 
the  same  place.  The  first  sensations  are  felt  about  two  or  three  hours  after 
inoculation ;  fever  and  general  indisposition  disappear  after  twenty-four  to 
thirty-six  hours ;  the  sensitiveness  and  oedema  last,  gradually  dying  away  in 


PROTECTIVE   INOCULATIONS.  305 

from  three  to  four  days.  The  symptoms  following-  the  second  inoculation 
were  generally  rather  more  marked,  but  of  shorter  duration.  The  whole 
recalls  the  sensation  of  a  bad  cold  in  the  head,  lasting  about  one  or  two  days. 

"The  microbes  introduced  under  the  skin  do  not  propagate,  but  after  a 
certain  time  they  die  and  disappear.  It  is  the  substances  which  they  contain, 
and  which  are  set  free  when  they  die,  that  act  upon  the  animal  organism  and 
confer  immunity  upon  it.  It  is  found  that  the  same  result  can  be  obtained  if 
the  microbe  be  killed  before  inoculation,  and  if  their  dead  bodies  only  be  in- 
jected. Thus  I  have  been  enabled  to  prepare  vaccines  preserved  in  weak 
solutions  of  carbolic  acid.  In  this  the  microbes  die  at  the  end  of  several 
hours,  and  the  vaccine  so  prepared  has  been  found  still  efficacious  six 
months  after  its  preparation.  It  is  evident  that  there  is  much  advantage  in 
this  state  of  preservation  of  the  microbes.  They  can  be  used  by  persons 
having  110  bacteriological  training,  and  the  absence  of  every  living  organism 
makes  them  perfectly  safe.  The  carbolic  acid  that  they  contain  preserves 
them  against  any  invasion  of  other  microbes.  Finally,  as  they  can  be  kept 
for  several  months,  their  preparation  can  be  entrusted  to  a  central  laboratory, 
whence  the  vaccine  ampoules  can  be  sent  out  to  operators.  But  it  may  be 
presumed  that  immunity  given  by  these  preserved  vaccines  will  not  equal  in 
persistency  that  produced  by  living  ones,  and  as  the  method  is  not  yet  backed 
up  by  established  statistics,  it  is  better  that  vaccinations  should  be  done  as 
much  as  possible  with  living  virus,  so  as  to  obtain  the  most  conclusive 
results. 

"As  to  the  length  of  time  that  immunity  produced  by  living  vaccine 
lasts,  we  have  not  yet  at  the  laboratory  animals  that  have  been  inoculated  at 
a  very  distant  date ;  those  upon  which  we  experimented  dated  from,  at  most, 
four  months  and  a  half.  At  the  end  of  this  time  their  immunity  was  found 
to  be  still  perfect,  and  we  do  not  despair  of  its  lasting  much  longer  yet. 

"  HARMLESSNESS   OF   THE   METHOD. 

"The  inoculations  upon  man,  added  to  the  hundreds  of  experiments  that 
we  have  made  upon  animals,  testify  to  the  perfect  harmlessness  of  these 
operations,  and  there  is  no  difficulty  in  proving  their  efficacy  by  experiment, 
no  matter  on  what  species  of  animal.  We  have  taken  twelve  guinea-pigs, 
and  vaccinated  six  of  them  with  vaccines  preserved  in  carbolic  acid  since 
September  8th  last.  Yesterday,  at  five  o'clock,  six  days  after  the  first  vacci- 
nation, we  injected  into  the  peritoneal  cavity  of  all  the  non- vaccinated  ani- 
mals a  fatal  dose  of  virus,  and  into  the  vaccinated  animals  we  injected  a 
double  dose.  The  six  vaccinated  animals  are  perfectly  well,  while  of  the 
others  two  have  already  died  of  choleraic  poisoning,  two  are  very  ill,  and  the 
others  will  certainly  soon  become  so.  But  it  is  evident  that  I  cannot  perform 
a  like  experiment  on  man  (but,  however,  this  would  be  the  only  means  of 
being  able  to  give  a  definite  experimental  demonstration)/' 

Further  details  as  to  the  method  are  given  by  Woodhead  in  the 
"Edinburgh  Hospital  Keports,"  as  follows: 

"In  order  to  be  absolutely  certain  that  the  virus  is  pure,  M.  Haffkine 
makes  cultivations  before  each  inoculation  of  the  human  subject,  by  Roux 
and  Yersin's  method,  one  devised  for  the  separation  of  the  diphtheria  bacillus. 
A  small  drop  of  the  virus  exalte  is  taken  on  a  spatula-shaped  needle,  and 
streak  after  streak  is  made  with  the  flat  of  this  needle  on  the  surface  of  the 
agar  in  the  tubes,  a  couple  of  tubes  being  used,  so  that  twelve  streaks  per- 
haps, in  all,  are  made  without  the  needle  being  recharged ;  in  the  earlier 
streaks,  of  course,  the  seed  bacilli  are  so  close  together  that  a  continuous  line 
of  colonies  makes  its  appearance  ;  but  along  the  course  of  the  later  streaks, 
colonies,  with  distinct  intervals  between  them,  are  developed  ;  part  of  one  of 
these  is  examined  under  the  microscope,  in  order  to  determine  that  it  is  made 
20 


306  PROTECTIVE   INOCULATIONS. 

up  only  of  comma  bacilli,  and  then  the  other  part  is  used  for  seed  material 
for  a  tube  culture  preparatory  to  inoculation. 

"The  inoculation  itself  is  an  exceedingly  simple  process  ;  the  needle  and 
the  syringe  are  boiled ;  the  tube  containing  the  material  to  be  used  for  inocu- 
lation receives  a  syringef ul  or  pipetteful  of  sterilized  beef  broth,  then  with  a 
platinum  needle  the  culture  is  thoroughly  mixed  with  this  broth,  so  that  a 
kind  of  emulsion  is  prepared;  this  emulsion  is  drawn  up  in  a  sterilized 
pipette,  and  is  then  passed  into  a  sterilized  conical  glass  covered  with  steril- 
ized paper.  If  a  sixth  of  the  culture  is  to  be  introduced,  two  more  syringe- 
fuls  or  pipettefuls  of  broth  are  to  be  added,  so  that  we  now  have  three  in  all  ; 
if  an  eighth,  three  are  added,  and  so  on  ;  the  whole  is  mixed,  and  then  half  a 
syringef  ul  is  taken  for  use  for  each  patient.  In  inoculating,  the  skin,  just 
above  the  crest  of  the  ilium,  is  thoroughly  cleansed  with  flve-per-ceiit  solu- 
tion of  carbolic  acid,  the  attenuated  virus  is  inoculated  011  the  left  side,  and 
then  after  an  interval  of  four  or  five  days  the  second  vaccine,  or  the  more 
virulent  form,  is  inoculated  on  the  right  side.  After  inoculation  everything 
that  has  been  used  is  thoroughly  boiled,  the  skin  of  the  patient  is  again 
washed  with  five-per-cent  carbolic  acid,  and  the  table  is  washed  down  with 
the  same  solution." 

Haffkine  commenced  his  experiments  on  man  by  inoculating  him- 
self, and  has  repeated  the  inoculation  three  times.  He  next  inoculated 
about  fifty  individuals  in  Paris,  Cherbourg,  and  Moscow,  and  demon- 
strated in  a  satisfactory  way  that  the  inoculations  are  without  danger. 

A  first  inoculation  in  an  unprotected  person  is  said  to  give  rise  to 
some  malaise  and  febrile  reaction,  to  pain  and  tumefaction  at  the 
point  of  inoculation,  and  swelling  of  the  neighboring  glands.  The 
second  inoculation  with  a  strong  virus,  made  after  an  interval  of  six 
days,  causes  also  some  elevation  of  temperature,  but  no  swelling  at 
the  point  of  inoculation.  This  slight  reaction  from  a  strong  virus  is 
supposed  to  be  satisfactory  evidence  of  a  certain  degree  of  immunity 
as  a  result  of  the  first  inoculation. 

The  results  of  the  protective  inoculations  by  Haffkine's  method, 
which  have  been  practised  in  India,  indicate  that  these  inoculations 
have  a  real  value,  but  that  immunity  is  not  immediately  established, 
and  consequently  that  during  an  epidemic  a  certain  number  of  fatal 
cases  may  be  expected  among  the  inoculated  as  well  as  among  the 
non-inoculated.  This  is  illustrated  by  the  results  of  inoculations 
made  among  the  prisoners  in  Gay  a  jail  (1894),  reported  by  Surgeon- 
Major  Macrae,  I.M.S.,  from  whose  report  we  quote  as  follows: 

"  Cholera  broke  out  in  Gaya  jail  on  the  9th  of  July,  and  from  that  date 
until  2d  August  34  cases  occurred,  with  20  deaths,  there  being  on  date  of  first 
attack  422  prisoners  in  jail.  The  disease  was  clearly  traceable  to  importa- 
tion, but  its  diffusion  among  the  prisoners  was  a  question  of  much  greater 
difficulty.  The  sanitary  condition  of  the  jail  is  excellent ;  it  was  built  quite 
recently,  on  the  latest  plans,  and  is  generally  considered  a  model  jail.  The 
water  supply,  which  is  from  a  well,  is  of  excellent  quality  and  protected 
from  pollution,  and  it  is  believed  that  the  spread  of  the  disease  was  largely 
due  to  the  agency  of  flies  finding  access  to  food  and  milk  after  being  in  con- 
tact with  cholera  poison,  and  contaminating  them.  From  the  9th  to  the  17th 
July  six  cases  occurred,  with  five  deaths. 


PROTECTIVE   INOCULATIONS. 


307 


"Many  of  the  prisoners  on  being  told  about  preventive  inoculation  wished 
to  be  inoculated,  and  M.  Haffkine,  who  had  previously  been  communicated 
with,  and  whose  zeal  and  enthusiasm  in  the  cause  that  he  so  well  advocates 
are  beyond  praise,  arrived  here  on  the  18th  July,  and  in  the  presence  of  Sur- 
geon-Colonel Harvey,  who  kindly  assisted,  and  myself,  inoculated  147 
prisoners,  and  on  the  19th  68,  making  a  total  of  215  out  of  433  present  in  the 
jail  on  that  date. 

"Being-  purely  voluntary,  no  selection  of  prisoners  was  possible  ;  but  all 
classes  in  the  jail  were  represented,  male  and  female,  old  and  young,  habit- 
uals  and  less  frequent  offenders,  strong  and  weakly,  convalescent  and  even 
hospital  patients  sent  their  representatives.  No  difference  of  any  kind  wTas 
made  between  inoculated  and  non-inoculated  prisoners ;  they  were  under 
absolutely  identical  conditions  as  regards  food,  water,  accommodation,  etc., 
in  short,  in  every  possible  respect. 

"  As,  owing  to  the  progress  of  the  epidemic,  a  large  number  of  prisoners 
were  removed  from  the  jail  into  camp,  it  will  be  found  convenient  to  con- 
sider the  effect  produced  by  the  anticholera  inoculation  under  three  head- 
ing^ : 

"(a)  TJie  first  will  include  the  period  from  the  18th  July,  the  date  of  first 
inoculations,  to  the  24th  July,  the  date  on  which  final  reinoc illations  were 
made,  and  refers  to  all  the  prisoners. 

"  (b)  Tlie  second  concerns  the  prisoners  who  remained  in  jail  after  the 
majority  were  removed  into  camp,  and  comprises  the  period  from  25th  July 
to  2d  August,  on  which  date  the  final  case  occurred  among  this  body  of 
prisoners. 

' '  (c)  The  third  refers  to  the  body  of  prisoners  who  were  moved  into  camp 
on  25th  July,  and  includes  the  period  between  that  date  and  1st  August, 
when  the  final  case  occurred  among  this  body. 


Average 
present. 

Chol- 
era. 

Percentage 
of  average 
strength. 

Deaths. 

Percentage 
of  aveniirc 
strength. 

Percentage 
of  deaths  to 
cases. 

Inoculated  

211.2 

5 

No.  1. 
2.37 

4 

1.89 

80.0 

Not  inoculated  

209.0 

7 

3.34 

5 

2.39 

71.42 

Inoculated  

32.5 

1 

No.  11. 
3.07 

Nil. 

Nil. 

Nil. 

Not  inoculated  

48.").-) 

7 

14.42 

3 

6.18 

42.86 

Inoculated    

171.42 

2 

No.  III. 
1.16 

1 

0.58 

50.0 

Not  inoculated  

146.5 

6 

4.09 

2 

1.36 

33.33 

"  The  conclusions  to  be  drawn  from  the  results  above  recorded  appear  to 
me  to  be  that  for  the  first  few  days  the  inoculations  have  scarcely  any  protec- 
tive influence  :  then  their  effect  seems  to  gradually  increase.  M.  Haffkine  in 
nis  publications  has  laid  stress  on  the  fact  that  he  anticipates  a  period  of  ten 
-days  would  elapse  from  date  of  first  inoculations  before  the  full  effect  would 
be  obtained. 


Inoculated 

Not  inoculated.  . 


DURING   THE  FIRST   FIVE 
DAYS  AFTER  FIRST  INOC- 
ULATION. 

FIRST  THREE   DAYS  AFTER 
SECOND  INOCULATION. 

LAST  six  DAYS. 

Cases. 

Deaths. 

Cases. 

Deaths. 

Cases. 

Deaths. 

5 

7 

4 
5 

3 
5 

1 
3 

Nil. 

8 

Nil. 

2 

308 


PROTECTIVE   INOCULATIONS. 


"  Further  observations  are  necessary  to  prove  whether  the  inoculations  as 
now  practised  will  prove  of  lasting1  benefit ;  the  results  obtained  in  Gaya  jail 
seem  to  me  to  justify  the  conclusion  that  their  temporary  beneficial  effect  is 
undoubted. 

"I  have  been  informed  by  M.  Haffkine  that  he  proposes  to  introduce  a 
certain  modification  of  his  method,  with  the  object  of  affording-  protection  to 
patients  during-  the  ten  days  necessary  for  the  action  of  his  vaccines.  I  think 
there  is  every  reason  to  believe  that  better  results  would  have  been  obtained 
here  had  the  inoculations  been  performed  at  an  earlier  period  instead  of  dur- 
ing- the  epidemic." 

In  a  paper  published  in  the  British  Medical  Journal  (January  26th, 
1895),  Haffkine  gives  the  following  summary  of  his  inoculations  in 
India : 

"  TABLE  SHOWING  THE  TOTAL  NUMBER  OF  PERSONS  ON  WHOM  OB- 
SERVATIONS HAVE  BEEN  MADE  IN  CALCUTTA,  GAYA,  CAWNPORE, 
AND  LUCKNOW. 


Number. 

Cases. 

Percentage 
of  Cases 
to  Strength. 

Deaths. 

Percentage 
of  Deaths 
to  Strength. 

Non  -inoculated  

1  735 

174 

10.63 

113 

6.51 

Inoculated  

500 

21 

4.20 

19 

3.80 

Total  

2,235 

195 

132 

Other  methods  of  producing  immunity  in  man  have  been  proposed, 
and  experiments  indicate  that  this  may  be  accomplished  through  the 
digestive  tract  by  the  ingestion  of  considerable  quantities  of  steril- 
ized cultures.  Thus  Klemperer  (1892)  has  obtained  results  which 
seem  to  show  that  immunity  in  man  may  be  induced,  not  only  by  the 
subcutaneous  injection  of  virulent  cultures,  but  also  by  the  subcuta- 
neous injection  of  the  milk  of  immunized  goats  and  by  the  ingestion  of 
cultures  sterilized  by  heat.  The  degree  of  immunity,  as  determined 
by  the  activity  of  the  blood  serum  of  the  immune  individual  for  the 
protection  of  guinea-pigs,  is  considerably  less,  however,  than  when 
repeated  injections  of  virulent  cultures  have  been  made.  The  blood 
serum  of  individuals  made  immune  by  the  last-mentioned  method  is 
said  by  Klernperer  to  protect  guinea-pigs  when  injected  into  the  cav- 
ity of  the  abdomen  in  the  dose  of  0.005  cubic  centimetre.  And  the 
injection  of  five  cubic  centimetres  of  milk  from  an  immunized  goat  is 
said  to  confer  such  an  immunity  that  0.25  cubic  centimetre  of  blood 
serum  from  the  immune  individual  is  sufficient  to  protect  a  guinea-pig 
from  cholera  cultures. 

Sawtschenko  and  Sabolotny  (1893),  as  a  result  of  a  series  of  ex- 
periments made  upon  themselves  and  laboratory  assistants,  arrive  at 
the  following  conclusions : 


PROTECTIVE   INOCULATIONS.  309 

"  1.  After  the  ingestion  of  sterilized  (by  heat)  and  subsequently  carbolized 
agar  cultures  of  cholera  bacteria  the  serum  of  man  acquires  an  immunizing 
property  as  regards  the  cholera  vibrio. 

4k2.  As  a  result  of  the  ingestion  of  sterilized  agar  cultures  the  individual 
is  protected  from  infection  with  virulent  cultures  of  the  cholera  vibrio  by  way 
of  the  intestine. 

"  3.  The  discharges  of  individuals  immune  against  cholera,  and  to  all  out- 
ward appearance  in  perfect  health,  may  contain  a  great  number  of  cholera 
vibrios  (in  case  they  are  in  any  way  introduced  into  the  intestine)  and  may 
thus  serve  to  propagate  the  malady." 

DIPHTHERIA. 

According  to  Roux  and  Yersin,  "  attenuated  varieties  "  of  the  diph- 
theria bacillus  may  be  obtained  by  cultivating  it  at  a  temperature  of 
39.5°  to  40°  C.  in  a  current  of  air;  and  these  authors  suggest  that  a 
similar  attenuation  of  pathogenic  power  may  occur  in  the  fauces  of 
convalescents  from  the  disease,  and  that  possibly  the  similar  non- 
pathogenic  bacilli  which  have  been  described  by  various  investigators 
have  originated  in  this  way  from  the  true  diphtheria  bacillus.  These 
authors  further  state,  in  favor  of  this  view,  that -from  diphtheritic 
false  membrane,  preserved  by  them  in  a  desiccated  condition  for  five 
months,  they  obtained  numerous  colonies  of  the  bacillus  in  question, 
but  that  the  cultures  were  destitute  of  pathogenic  virulence.  They 
say : 

"  It  is  then  possible,  by  commencing  with  a  virulent  bacillus  of  diphtheria, 
to  obtain  artificially  a  bacillus  without  virulence,  quite  similar  to  the  attenu- 
ated bacilli  which  may  be  obtained  from  a  benign  diphtheritic  angina,  or  even 
from  the  mouth  of  certain  persons  in  good  health.  This  microbe,  obtained 
artificially,  resembles  completely  the  pseudo-diphtheritic  bacillus  ;  like  it,  it 
grows  more  abundantly  at  a  low  temperature  ;  it  renders  bouillon  more  rap- 
idly alkaline  ;  it  grows  with  difficulty  in  the  absence  of  oxygen." 

Subcutaneous  inoculations  in  guinea-pigs  of  a  small  quantity  of  a 
pure  culture  of  the  bacillus  (0.1  to  0.5  cubic  centimetre  of  a  bouillon 
culture)  cause  death  in  from  one  to  four  or  five  days.  The  usual 
changes  observed  at  the  autopsy  are — 

"  An  extensive  local  oedema^  with  more  or  less  hyperamiia  and  ecchymo- 
wis  at  the  site  of  inoculation,  frequently  swollen  and  reddened  lymphatic 
glands,  increased  serous  fluid  in  the  peritoneum,  pleura,  and  pericardium, 
enlarged  and  hemorrhagic  suprarenal  capsules,  occasionally  slightly  swollen 
spleen,  sometimes  fattA  degenerations  in  the  liver,  kidney,  and  myocardium. 
We  have  always  found  the  Loftier  bacilli  at  the  seat  of  inoculation  most 
abundant  in  a  grayish- white,  fibrino-purulent  exudate  present  at  the  point  of 
inoculation,  and  becoming  fewer  at  a  distance  from  this,  so  that  the  more  re- 
mote parts  of  the  oedematous  fluid  do  not  contain  any  bacilli "  (Welch  and 
Abbott). 

The  authors  quoted  agree  with  Loffler  and  others  in  stating  that 
the  bacillus  is  found  only  at  the  point  of  inoculation.  In  all  cases 


310  PROTECTIVE   INOCULATIONS. 

their  cultures  from  the  blood  and  from  the  various  organs  gave  a 
negative  result. 

Rabbits  are  not  so  susceptible,  and  may  recover  after  the  subcu- 
taneous inoculation  of  very  small  doses,  but  usually  die  in  from  four 
to  twenty  days  when  two  to  four  cubic  centimetres  of  a  bouillon  cul- 
ture have  been  introduced  beneath  the  skin.  In  these  animals,  also, 
there  is  an  extensive  local  oedema,  an  enlargement  of  the  neighboring 
lymphatic  glands,  and  a  fatty  degeneration  of  the  liver.  Eoux  and 
Yersiu  have  shown  that  in  rabbits,  when  death  does  not  ensue  too 
quickly,  paralysis  of  the  posterior  extremities  frequently  occurs,  thus 
completing  the  experimental  proof  of  the  specific  pathogenic  power 
of  pure  cultures  of  this  bacillus. 

Similar  symptoms  are  produced  in  pigeons  by  the  subcutaneous 
inoculation  of  0.5  cubic  centimetre  or  more,  but  they  commonly  re- 
cover when  the  quantity  is  reduced  to  0.2  cubic  centimetre  (Roux  and 
Yersin) . 

The  rat  and  the  mouse  have  a  remarkable  immunity  from  the  effects 
of  this  poison.  Thus,  according  to  Roux  and  Yersin,  a  dose  of  two 
cubic  centimetres,  which  would  kill  in  sixty  hours  a  rabbit  weighing 
three  kilogrammes,  is  without  effect  upon  a  mouse  which  weighs  only 
ten  grammes. 

Old  cultures  are  somewhat  less  virulent  than  fresh  ones,  but  when 
replanted  in  a  fresh  culture  medium  they  manifest  their  original  viru- 
lence. Thus  a  culture  upon  blood  serum  which  was  five  months  old 
was  found  by  Roux  and  Yrersin  to  kill  a  guinea-pig  in  five  days,  but 
when  replanted  it  killed  a  second  animal  of  the  same  species  in 
twenty -four  hours. 

Evidently  a  microorganism  which  destroys  the  life  of  a  susceptible 
animal  when  injected  beneath  its  skin  in  small  quantity,  and  which 
nevertheless  is  only  found  in  the  vicinity  of  the  point  of  inoculation, 
must  owe  its  pathogenic  power  to  the  formation  of  some  potent  toxic 
substance,  which,  being  absorbed,  gives  rise  to  toxaemia  and  death. 
This  inference  in  the  case  of  the  diphtheria  bacillus  is  fully  sustained 
by  the  results  of  experimental  investigations.  Roux  and  Yersiu 
(1888)  first  demonstrated  the  pathogenic  power  of  cultures  which 
had  been  filtered  through  porous  porcelain.  Old  cultures  were  found 
by  these  experimenters  to  contain  more  of  the  toxic  substance  than 
recent  ones,  and  to  cause  the  death  of  a  guinea-pig  in  a  dose  of  two 
cubic  centimetres  in  less  than  twenty -four  hours.  The  filtered  cul- 
tures produced  in  these  animals  the  same  effects  as  those  containing 
the  bacilli — local  oedema,  hemorrhagic  congestion  of  the  organs, 
effusion  into  the  pleural  cavity.  Somewhat  larger  doses  were  fatal 


PROTECTIVE   INOCULATIONS. 

to  rabbits,  and  a  few  drops  injected  subcutaneously  sufficed  to  kill  a 
small  bird  within  a  few  hours.  In  their  second  paper  (1889)  the 
authors  mentioned  state  that  so  long  as  the  reaction  of  a  culture  in 
bouillon  is  acid  its  toxic  power  is  comparatively  slight,  but  that  in 
old  cultures  the  reaction  is  alkaline,  and  in  these  the  toxic  potency  is 
greatly  augmented.  With  such  a  culture,  filtered  after  having  been 
kept  for  thirty  days,  a  dose  of  one-eighth  of  a  cubic  centimetre  in- 
jected subcutaneously,  sufficed  to  kill  a  guinea-pig;  and  in  larger 
amounts  it  proved  to  be  fatal  to  dogs  when  injected  directly  into  the 
circulation  through  a  vein. 

The  same  authors,  in  discussing  the  nature  of  the  poison  in  their 
filtered  cultures,  infer  that  it  is  related  to  the  diastases,  and  state  that 
its  toxic  potency  is  very  much  reduced  by  exposure  to  a  comparatively 
low  temperature— 58°  C.,  for  two  hours — and  completely  destroyed  by 
the  boiling  temperature — 100C  C.,  for  twenty  minutes.  It  was  found 
to  be  insoluble  in  alcohol,  and  the  precipitate  obtained  by  adding  al- 
cohol to  an  old  culture  proved  to  contain  the  toxic  substance.  Loffler 
also  has  obtained,  by  adding  five  volumes  of  alcohol  to  one  of  a  pure 
culture,  a  white  precipitate,  soluble  in  water,  which  killed  rabbits  in 
the  dose  of  0.1  to  0.2  gramme  when  injected  beneath  the  skin  of  these 
animals.  It  gave  rise  to  a  local  oedema  and  necrosis  of  the  skin  in 
the  vicinity  of  the  point  of  inoculation,  and  to  hyperaemia  of  the  in- 
ternal organs.  This  deadly  toxin  appears  to  be  an  albuminoid  sub- 
stance, but  its  exact  chemical  composition  has  not  yet  been  deter- 
mined. 

Brieger  and  Frankel  (1891)  obtained  results  corresponding  with 
those  previously  reported  by  Roux  and  Yersin.  Their  researches 
showed  that  the  toxic  substance  contained  in  diphtheria  cultures  is 
destroyed  by  a  temperature  of  60°  C. ;  that  it  is  soluble  in  water,  and 
insoluble  in  alcohol;  that  it  does  not  pass  through  a  dialyzing  mem- 
brane, and  has  not  the  chemical  characters  of  the  ptomaines  or  toxins, 
but  is  an  albuminous  body — a  toxalbumin.  It  wras  obtained  by  the 
authors  named  by  precipitation  with  slightly  acidified  (acetic  acid) 
alcohol;  the  precipitate,  after  being  washed  in  a  dialyzer  and  dried 
in  a  vacuum  at  a  temperature  of  40°  C.,  was  a  snow-white,  amorphous, 
crumbling  mass. 

Wassermann  and  Proskauer  (1892)  found  that  the  alcoholic  pre- 
cipitate from  diphtheria  cultures  contains  two  different  substances, 
which  are  distinguished  by  their  different  degrees  of  solubility  in 
dilute  and  absolute  alcohol ;  both,  however,  give  the  usual  reactions 
of  albuminous  bodies,  and  pass  very  slowly  through  a  dialyzing  mem- 
brane. Only  one  of  these  substances  possesses  toxic  properties. 


312  PROTECTIVE   INOCULATIONS. 

After  the  removal  of  peptone  and  globulin  from  the  filtered  cultures, 
these  were  evaporated  and  a  precipitate  obtained  of  one  of  the 
albuminous  substances  by  means  of  sixty  to  seventy  per  cent  al- 
cohol. The  other  substance  remained  in  solution,  and  was  sub- 
sequently obtained  by  precipitation  with  absolute  alcohol.  The 
substance  first  obtained  by  this  method  is  toxic,  and  the  other  pre- 
cipitate is  not.  The  authors  named  succeeded  in  killing  rabbits  with 
the  toxalbumin  obtained  in  this  way,  but  were  not  able  to  produce 
immunity  in  these  animals  by  the  injection  of  non-fatal  doses.  Friin- 
kel  (1891)  had  previously  reported  his  failure  to  immunize  guinea-pigs 
by  the  injection  of  the  dry  precipitate,  obtained  in  his  experiments 
from  diphtheria  cultures;  but  when  filtered  cultures,  or  cultures 
sterilized  by  heat  (55°  C.  for  one  hour),  were  injected  into  these  ani- 
mals, they  showed  an  increased  resistance  to  the  pathogenic  action  of 
virulent  cultures.  Still  better  results  were  obtained  when  ten  cubic 
centimetres  of  a  bouillon  culture,  heated  to  100°  C.,  were  injected 
subcutaneously,  but  still  this  method  was  not  entirely  reliable.  But 
true  immunity  was  established  by  injecting  into  the  peritoneal  cavity 
ten  to  twenty  cubic  centimetres  of  a  bouillon  culture  heated  to  from  65° 
to  70°  C.  for  one  hour.  The  immunity  was  not  fully  established  until 
about  fourteen  days  after  the  protective  inoculation.  Friinkel  arrives 
at  the  conclusion  that  the  cultures  must  contain  an  immunizing  sub- 
stance as  well  as  a  toxic  proteid,  as  the  diphtheria  toxalbumin  is  de- 
stroyed by  the  temperature  (65°  to  70°  C.)  used  in  the  preparation  of 
his  cultures  for  producing  immunity. 

Behriug,  in  the  same  year  (1891),  commenced  his  experiments 
upon  diphtheria  immunity.  Guinea-pigs  were  made  immune  by  the 
use  of  sterilized  cultures,  and  by  inoculations  with  virulent  cultures, 
four  weeks  old,  to  which  iodine  terchloride  had  been  added  in  the 
proportion  of  1 : 500— the  mixture  was  allowed  to  stand  for  sixteen 
hours.  Animals  were  also  immunized  by  injecting  beneath  the  skin 
a  virulent  culture  of  the  bacillus,  and  then  treating  them  with  sub- 
cutaneous injections  of  iodine  terchloride  (two  cubic  centimetres), 
which  was  thrown  under  the  skin  for  three  days  in  succession  in  the 
vicinity  of  the  point  of  inoculation.  The  guinea-pigs  treated  in  this 
way  remained  sick  for  some  time,  but  finally  recovered  and  were 
subsequently  immune.  Still  better  results  were  obtained  when  rab- 
bits were  subjected  to  the  same  treatment.  The  animals  were  im- 
mune against  the  toxic  action  of  sterilized  cultures,  as  well  as  against 
infection  by  virulent  diphtheria  bacilli. 

In  subsequent  experiments  (1892)  Behring  and  Wernicke  used 
cultures  which  had  been  attenuated  bv  contact  with  iodine  terchloride 


PROTECTIVE   INOCULATIONS.  313 

for  from  thirty-six  to  forty-eight  hours,  and  proved  that  the  method 
could  be  successfully  employed  in  immunizing  sheep ;  and  the  fact 
was  ascertained  that  blood  serum  from  an  immune  animal  could  be 
used  with  success  in  arresting  diphtheritic  infection  in  susceptible 
animals.  To  preserve  the  serum,  which  they  obtained  from  immun- 
ized sheep,  rabbits,  and  guinea-pigs,  they  added  to  it  0.5  per  cent  of 
pure  carbolic  acid.  For  producing  immunity  they  found  that  a 
smaller  amount  of  serum  was  required  than  wras  necessary  for  the 
cure  of  an  animal  already  infected.  If  the  injection  was  made  imme- 
diately after  infection,  from  one  and  a  half  to  two  times  the  amount 
was  required;  eight  hours  after  infection  the  amount  was  three  times 
as  great,  and  twenty-four  to  thirty-six  hours  after  infection  the  dose 
required  was  eight  times  the  immunizing  dose. 

The  immunizing  value  of  blood  serum  from  different  animals  was 
estimated  by  finding  the  smallest  dose  which  would  protect  an  animal 
from  fatal  infection  by  the  minimum  lethal  dose  of  a  culture  the  toxic 
potency  of  which  had  been  carefully  determined.  The  value  is  ex- 
pressed in  figures  which  give  the  proportion  required  compared  with 
the  body  weight  of  the  animal.  Thus  an  immunizing  value  of  one 
hundred  would  mean  that  one  gramme  of  the  serum  is  sufficient  to 
protect  an  animal  weighing  one  hundred  grammes  from  the  fatal 
effect  which  would  be  produced  in  a  control  animal  of  the  same  weight 
by  infection  with  a  virulent  culture  of  the  diphtheria  bacillus  in  the 
minimum  doses  required  to  produce  this  result.  The  cultures  em- 
ployed are  made  in  bouillon  containing  one  per  cent  of  peptone ;  they 
are  inoculated  from  agar  cultures  and  are  kept  in  the  incubating  oven 
for  two  days.  Cultures  prepared  in  this  way  were  found  to  be  quite 
uniform  in  their  pathogenic  virulence  as  tested  upon  guinea-pigs.  But 
when  cultures  are  kept  for  some  time  there  is  an  increase  in  virulence. 
Thus  a  culture  obtained  from  a  fatal  case  of  diphtheria  which  in  1890 
killed  guinea-pigs  in  three  to  four  days,  when  injected  subcutaneously 
in  the  dose  of  0.1  cubic  centimetre  (two-days-old  bouillon  culture), 
at  the  end  of  a  year  was  fatal  to  these  animals  in  the  dose  of  0.025 
cubic  centimetre.  This  increase  in  virulence  is  ascribed  to  the  fact 
that  the  cultures  were  renewed  at  long  intervals. 

More  recently  (1894)  Behring  has  fixed  a  standard  for  what  he 
calls  a  normal  therapeutic  serum.  This  is  a  serum  which  when  in- 
jected into  guinea-pigs  in  the  proportion  of  1 : 5,000  of  bod}'  weight 
saves  the  animal  from  the  fatal  effects  of  ten  times  the  minimum  dose 
of  a  culture  in  bouillon,  two  days  old,  which  would  kill  a  control  ani- 
mal not  treated. 

In  a  subsequent  communication  (November,  1894)  Behring  states 


314  PROTECTIVE   INOCULATIONS. 

his  conclusion  that  for  producing  immunity  in  man,  one  hundred  and 
fifty  normal  antitoxin  units  should  be  given,  instead  of  sixty  as  he 
had  previously  recommended. 

The  serum  manufactured  under  his  direction  is  said  (September, 
1894)  to  be  of  two  kinds — one,  obtained  from  the  horse,  has  a  value 
of  sixty  normal  antitoxin  units ;  the  other  has  a  value  of  one  hundred 
and  forty  units.  Of  the  weaker  serum  Behring  says  experience  has 
demonstrated  that  for  children  under  ten  years  of  age  ten  cubic  cen- 
timetres is  sufficient  to  arrest  the  progress  of  the  disease  and  effect  a 
cure  if  given  within  two  or  three  days  from  the  outset  of  the  attack. 
For  producing  immunity  in  children  subject  to  infection,  one-tenth 
of  this  amount  (one  cubic  centimetre)  is  said  to  be  sufficient.  Of 
the  stronger  serum  one  cubic  centimetre  is  sufficient  to  arrest  the  dis- 
ease during  the  incubation  period ;  and,  according  to  Behring,  out  of 
one  hundred  cases  treated  during  the  first  forty-eight  hours  with  a 
single  therapeutic  dose  (ten  cubic  centimetres  of  serum  having  a  value 
of  sixty,  equals  six  hundred  normal  units),  not  five  will  die.  The 
later  the  treatment  is  commenced  the  larger  will  be  the  dose  required. 
Behring  further  states  that  the  diphtheria  antitoxin  has  no  injurious 
effect  upon  animals  in  the  largest  doses  that  have  been  employed, 
and  that  aside  from  its  antitoxic  power  its  properties  are  entirely 
negative  so  far  as  living  animals  are  concerned. 

Aronson  (1893),  in  experiments  on  dogs,  succeeded  in  producing 
immunity  by  the  use  of  attenuated  cultures,  or  of  cultures  to  which 
formaldehyde  had  been  added ;  also  by  feeding  the  animal  large  quan- 
tities of  diphtheria  bouillon;  and,  finally,  by  injection  of  the  blood  of 
naturally  immune  animals  (white  rats)  into  which  large  quantities  (ten 
cubic  centimetres)  of  a  virulent  culture  had  been  injected.  Two  months 
after  receiving  several  such  injections  it  was  found  that  0.2  gramme 
of  blood-serum  from  the  rat  sufficed  to  save  a  guinea-pig  from  fatal 
infection.  In  experiments  on  dogs  an  immunity  was  established  in 
six  weeks  by  the  injection  of  a  large  amount  of  a  virulent  culture.  Its 
serum  had  a  value  of  1 : 30,000,  i.e.,  0.01  cubic  centimetre  of  this  serum 
sufficed  to  protect  a  guinea-pig  weighing  three  hundred  grammes. 
From  one  hundred  grammes  of  this  serum  Aronson  claims  to  have  ob- 
tained 0.8  gramme  of  a  substance  which  had  a  value  of  1 : 500,000,  as 
tested  in  the  treatment  of  an  animal  which  had  received  ten  times 
the  minimum  fatal  dose  of  a  two-days'  bouillon  culture.  A  ten-per- 
cent solution  of  this  substance  had,  therefore,  ten  times  the  value  of 
Behring.' s  "  normal  serum. "  The  precipitated  antitoxin  was  soluble  in 
water,  and  more  readily  so  in  a  slightly  alkaline  solution,  and  gave 
all  the  reactions  of  an  albuminous  body.  When  dried  in  vacuo  at 


PROTECTIVE    INOCULATIONS.  315 

40°  C.,    and  then  heated  to  102°  C.,  it  still  retained  its   antitoxic 
potency. 

Ehrlich,  Kossel,  and  Wasserniann  (1894)  have  made  experiments 
upon  goats,  which  they  found  very  susceptible  to  the  action  of  the 
diphtheria  poison.  Sterilized  cultures  were  first  injected  in  gradually 
increasing  amounts,  and  later  virulent  cultures.  In  this  way  they  ob- 
tained a  serum  which  has  a  value  sixty  times  that  of  Behriug's  "nor- 
mal serum."  In  a  subsequent  communication  (1894)  Wassermann 
gives  an  account  of  his  experiments  with  the  milk  of  immunized 
goats,  which  contains  the  antitoxin  in  considerable  quantity,  and 
from  which  it  was  obtained  in  a  concentrated  form  by  the  following 
method :  The  milk  is  obtained  in  sterilized  vessels  and  twenty  cubic 
centimetres  of  normal  hydrochloric  acid  are  added  to  each  litre;  a,t 
sufficient  quantity  of  rennet  is  then  added  to  coagulate  the  casein, 
and  this  is  separated  from  the  liquid,  which  is  then  shaken  up  with 
chloroform  for  some  time.  The  liquid  is  now  allowed  to  stand  in 
order  that  the  butter,  which  has  been  dissolved  by  the  chloroform, 
may  sink  to  the  bottom.  The  clear  liquid  is  then  decanted  and  the 
antitoxin  precipitated  from  it  by  means  of  ammonium  sulphate  (thirty 
to  thirty-three  per  cent).  The  precipitate  is  rapidly  dried  upon 
porous  porcelain  plates,  in  vacuo,  and  then  dissolved  in  water  in  the 
proportion  of  ten  parts  for  one  hundred  of  milk  first  employed — a 
concentration  to  one-tenth.  Of  this  solution  0.125  cubic  centimetre 
was  found  to  neutralize  0.9  cubic  centimetre  of  a  toxin  which  killed 
guinea-pigs  weighing  five  hundred  grammes  in  the  dose  of  0.1  cubic 
centimetre.  This  toxin  was  an  old  bouillon  culture  of  the  diphtheria 
bacillus  to  which  0.5  per  cent  of  carbolic  acid  had  been  added  to 
preserve  it.  In  a  communication  of  the  same  date  Ehrlich  and  Was- 
sermann report  that  they  have  for  some  time  had  a  cow  immunized 
to  such  a  degree  that  one  cubic  centimetre  of  its  milk  protects  guinea- 
pigs  from  the  fatal  effects  of  0.9  cubic  centimetre  of  the  above-men- 
tioned toxin.  The  antitoxic  value  of  the  milk  of  an  immunized  cow 
or  goat,  as  compared  with  that  of  its  blood,  is  estimated  by  Ehrlich 
and  Wassermann  as  from  1 : 15  to  1 : 30 — usually  about  1 : 20. 

Aronson,  in  testing  his  antitoxin,  uses  a  bouillon  culture  of  the 
diphtheria  bacillus  two  and  one-half  months  old,  which  he  preserves 
by  the  addition  of  0.3  per  cent  of  trikresol.  He  finds  that  the  im- 
munity which  results  from  injections  of  the  antitoxin  is  established 
at  once;  that  it  is  not  accompanied  by  any  reaction  or  symptom  of 
sickness ;  and  that  it  is  of  comparatively  short  duration. 

As  a  result  of  extended  experiments  made  at  the  Pasteur  Institute 
in  Paris,  Koux  has  perfected  the  following  method  for  the  production 


316  PROTECTIVE   INOCULATIONS. 

of  an  antitoxin  suitable  for  use  in  the  treatment  of  diphtheria  in  man. 
The  horse  has  been  found  the  most  suitable  animal  for  this  purpose, 
on  account  of  his  slight  susceptibility  and  the  ease  with  which  a  high 
degree  of  immunity  can  be  established;  and  because  of  the  large 
amount  of  blood  that  may  be  drawn  without  injury  to  the  animal. 
Roux  prepares  his  toxin  by  cultivating  the  diphtheria  bacillus  in  a 
slightly  alkaline  bouillon  made  from  beef  and  containing  two  per 
cent  of  peptone  and  0.5  per  cent  of  sodium  chloride.  This  medium 
is  placed  in  flat-bottomed  flasks,  and  should  not  be  more  than  half  an 
inch  in  depth.  Two  glass  tubes  pass  into  the  flask,  which  serve  for 
inlet  and  outlet  tubes  to  be  used  in  passing  a  current  of  air  over  the 
cultures.  This  is  commenced  when  the  growth  is  fairly  started,  at 
the  end  of  twenty-four  hours,  and  the  air  should  be  moist  to  prevent 
the  evaporation  of  the  culture.  In  Roux's  laboratory  a  flask  is  used 
which  has  a  tube  attached  to  one  side,  about  an  inch  from  the  bottom, 
and  which  is  known  as  a  Fernback  flask.  A  flocculent  deposit  falls 
to  the  bottom  and  gradually  accumulates  for  about  a  month.  This 
consists  of  bacilli  which  have  for  the  most  part  lost  their  vitality  and 
are  undergoing ,  degeneration.  At  the  end  of  thirty  days,  during 
which  time  they  are  kept  in  an  incubating  oven  at  a  temperature  of 
37°  C.,  the  cultures  are  passed  through  a  Pasteur-Chamberland  filter, 
and  0.5  per  cent  of  carbolic  acid  may  be  added  in  order  to  preserve 
them.  This  filtrate  is  so  toxic  that  a  dose  of  0.1  cubic  centimetre 
will  kill  a  guinea-pig  weighing  five  hundred  grammes  in  less  than 
forty-eight  hours.  A  healthy  horse  is  selected  and  receives  at  first  a 
dose  of  0.5  cubic  centimetre  of  the  filtered  culture  (or  of  the  clear 
fluid  obtained  from  a  culture  by  decantation,  and  containing  0.5  per 
cent  of  carbolic  acid).  The  dose  is  gradually  increased  at  intervals 
of  a  few  days,  and  is  followed  each  time  by  some  febrile  reaction  and 
tumefaction  at  the  point  of  inoculation.  When  the  reaction  is  exces- 
sive, a  little  Gram's  solution  is  added  to  the  following  dose.  The 
usual  plan  of  treatment  is  stated  by  Kinyoun  as  follows : 

"  First  day,  1  to  2  c.c.  of  pure  toxins,  of  which  1  to  10  c.c.  fatal  to  a  500- 
gm.  guinea-pig  ;  eighth  day,  1  c.c.  :  fourteenth  day,  1.5  c.c.  :  twentieth  day, 
2  c.c.  ;  twenty-eighth  day,  3  c.c.  ;  thirty-third  day,  5  c.c.  ;  thirty-eighth  day, 
8  c.c.  ;  forty- third  day,  10  c.c.  ;  forty-seventh  day,  20  c.c. ;  fifty-first  day,  30 
c.c.  ;  fifty-sixth  day,  50  c.c.  ;  sixty-second  day,  50  c.c.  ;  sixty-eighth  day,  60 
c.c.  ;  seventy-fourth  day,  100  c.c.  ;  eightieth  day,  250  c.c.  ;  eighty-eighth  clay, 
250  c.c. 

"When  the  first  injections  are  given  there  is  quite  a  marked  local  and  gen- 
eral reaction  to  the  poison  ;  there  is  an  oedema  at  the  point  of  the  injection, 
which  is  followed  by  a  distinct  inflammatory  process— hard  in  the  centre  and 
soft  and  cedematous  at  its  periphery.  The  general  reaction  is  manifested  by 
a  rise  in  the  temperature,  1°  to  2°  C.,  loss  of  appetite,  and  occasionally  cramps. 
The  reaction  must  be  taken  as  the  guide  in  the  future  dosage,  and  a  sufficient 


PROTECTIVE   INOCULATIONS.  317 

time  must  be  allowed  to  elapse  between  the  injections  for  the  complete  recov- 
ery from  the  general  and  local  effects.  As  the  quantity  of  the  toxins  is  in- 
creased the  general  effects  generally  decrease,  perhaps  a  rise  of  a  degree  for 
twenty-four  hours.  The  local  effect  partakes  more  of  an  cedenia,  and  has  the 
character  of  an  inflammation. 

"  At  a  certain  stage,  usually  after  two  months'  treatment,  when  fifty  to 
sixty  cubic  centimetres  can  be  injected  without  harm,  there  is  no  general  re- 
action, but  a  large  oedema  at  the  site  of  the  injection,  which  disappears 
within  from  twenty -four  to  forty-eight  hours.  Toward  the  last,  even  when 
two  hundred  to  three  hundred  cubic  centimetres  are  given,  there  is  only  an 
enormous  oedema,  which  disappears  within  from  twelve  to  eighteen  hours. 
When  these  inordinately  large  quantities  can  be  given  with  only  a  local  re- 
action being  manifest,  the  horse  has  come  well  under  the  influence,  and  the 
blood  will  be  found  to  be  rich  in  the  antitoxin. 

"There  is  a  curious  fact  well  worth  noting  :  At  the  end  of  the  second 
month  of  treatment,  when  the  horse  can  bear  as  much  as  fifty  to  sixty  cubic 
centimetres  of  the  toxins  without  discomfort,  the  blood  will  be  found  to  con- 
tain but  little  of  the  antitoxin.  The  antitoxin  only  appears  after  repeated 
stimulation  of  the  cells  (?)  by  the  large  and  frequent  doses  of  the  toxins." 

The  subcutaneous  injections  do  not  yield  a  serum  as  rich  in  the 
antitoxins  as  when  the  toxins  are  injected  directly  into  the  blood  cur- 
rent. When  it  is  desired  to  do  this,  toward  the  last  of  the  treatment 
the  toxins  are  injected  directly  into  the  jugular  vein.  The  process  is 
tedious  and  requires  a  longer  time,  and  for  practical  purposes  has  not 
been  found  so  satisfactory  as  the  simple  subcutaneous  injection.  The 
strength  of  the  serum  is  tested  by  using  young  guinea-pigs  of  five 
hundred  grammes  weight.  One  gramme  of  the  serum  usually  will 
protect  fifty  thousand  grammes  of  guinea-pig  against  a  fresh  virulent 
culture  of  the  Bacillus  diphtheria.  This  is  the  strength  that  is  used 
in  the  hospitals.  By  the  intravenous  injections  a  serum  of  the  pro- 
tective strength  of  1 : 100,000  can  be  obtained. 

When  fully  immunized  from  six  to  eight  litres  of  blood  may  be 
taken  from  a  horse  at  one  time,  but  as  a  rule  it  is  better  not  to  take 
more  than  three.  The  blood  is  drawn  from  the  jugular  vein,  by  means 
of  a  small  trocar  and  cannula,  into  wide-mouthed  bottles  having  a  ca- 
pacity of  2.5  litres;  these  are  placed  in  an  ice  chest  for  twenty-four 
hours  to  give  time  for  the  separation  of  the  serum,  which  is  then 
transferred  to  smaller  receptacles  for  preservation. 

The  dose  of  serum  prepared  in  this  way,  when  used  to  protect  from 
diphtheria  infection,  is  five  cubic  centimetres  for  a  child  under  ten 
years  of  age,  and  ten  cubic  centimetres  for  older  children.  This  does 
not  afford  an  absolute  protection,  but  is  believed  to  be  generally 
effective,  and  in  case  of  failure  the  attack  is  said  to  be  of  a  mild 
character.  The  curative  dose  of  Roux's  serum  is  twenty  cubic  centi- 
metres for  children,  and  thirty  to  forty  cubic  centimetres  for  patients 
over  fifteen  years  of  age.  The  larger  dose  is  divided  and  given,  at 
the  same  time,  by  subcutaneous  injection  in  two  places.  Antiseptic 


318  PROTECTIVE    INOCULATIONS. 

precautions  are  taken  in  giving  these  injections,  and  a  little  absorbent 
cotton  is  placed  over  the  puncture. 

FOOT-AND-MOUTH   DISEASE. 

This  is  an  infectious  disease  of  cattle,  sheep,  goats,  and  swine,  the 
etiology  of  which,  so  far  as  the  specific  infectious  agent  is  concerned, 
has  not  been  determined. 

The  extent  to  which  the  disease  in  question  prevails  in  some  parts 
of  Europe  is  shown  by  the  statistics  for  1891  of  the  prevalence  of  this 
disease  in  Germany.  According  to  the  Eeichsseuchenbericht  it  pre- 
vailed most  extensively  in  the  southern  portion  of  Germany.  The 
total  number  of  infected  farms  was  47,865;  the  total  number  of  in- 
fected cattle  was  394,640;  of  sheep,  240,904;  of  goats,  3,378;  of  swine, 
182,208.  Behla  (1892)  has  made  inoculation  experiments  with  the 
filtered  saliva  of  infected  cattle  to  which  he  added  one  to  two  per  cent 
of  carbolic  acid,  and  claims  to  have  produced  immunity  in  young  pigs 
and  lambs.  The  duration  is  not,  however,  very  long  even  in  animals 
which  have  recovered  from  an  attack  of  the  disease — said  to  be  from 
six  months  to  three  years — and  a  practical  method  of  restricting  the 
disease  by  means  of  protective  inoculations  has  not  as  yet  been  intro- 
duced. 

GLANDERS. 

The  toxic  substances  produced  in  cultures  of  the  glanders  bacillus 
when  concentrated  in  the  form  of  a  glycerin  extract  constitute  the  so- 
called  mallein,  which  has  been  extensively  used  in  the  diagnosis  of 
glanders  in  horses.  As  is  the  case  when  animals  infected  with  tuber- 
culosis are  inoculated  with  tuberculin,  animals  infected  with  glanders 
have  a  decided  rise  of  temperature  after  receiving  a  sufficient  dose  of 
mallein  beneath  the  skin. 

Babes  (1892)  reports  that  the  toxic  substance  in  cultures  of  the 
glanders  bacillus  may  be  obtained  by  precipitation  with  alcohol;  and 
that  mallein  obtained  from  filtered  cultures  to  which  glycerin  has 
been  added,  or  the  alcoholic  precipitate,  may  be  successfully  used 
for  protecting  susceptible  animals  against  glanders  infection  or  for 
curing  the  disease  after  infection.  He  has  demonstrated  the  thera- 
peutic value  upon  guinea-pigs  and  upon  two  horses  which  are  said  to 
have  been  cured  of  chronic  glanders.  When  large  and  repeated  doses 
are  injected  into  healthy  animals  they  produce  nephritis  and  general 
marasmus.  The  action  upon  horses  infected  with  glanders  is  very 
marked  and  small  doses  may  even  cause  death. 

Kresling  (1892)  recommends  potato  cultures  as  preferable  to  bouil- 


PROTECTIVE   INOCULATIONS.  319 

Ion  cultures  for  the  preparation  of  mallein.  The  potatoes  are  to  be 
washed,  before  sterilization,  in  a  five-per-cent  bicarbonate  of  soda 
solution,  "until  the  wash-water  remains  clear."  They  are  then 
cooked  for  an  hour  and  twenty  minutes.  After  planting  upon  the 
surface  glanders  bacilli  from  a  previous  culture  they  are  placed  in  an 
incubator  at  36°  to  36.5°  C.,  with  provision  to  prevent  them  from  be- 
coming dry.  At  the  end  of  two  weeks  the  growth  is  removed  with  a 
platinum  spatula  and  added  to  nine  parts  of  water,  in  which  it  is  well 
mixed  by  rubbing.  It  is  then  allowed  to  stand  for  twenty-four  hours, 
after  which  it  is  sterilized  for  fifteen  minutes  at  110°  0.  (a  lower  tem- 
perature would  no  doubt  answer  quite  as  well).  After  cooling  it  is 
passed  through  a  Chamberlain  filter  by  means  of  a  pressure  of  six 
atmospheres.  The  filtrate  is  then  carefully  evaporated  over  a  water- 
bath  to  one-fourth  its  volume,  and  to  this  concentrated  extract  gly- 
cerin is  added  in  the  proportion  of  one  part  to  two.  The  mixture  is 
again  sterilized  in  the  autoclave  at  110°  C.  When  injected  into 
healthy  horses  in  the  dose  of  two  cubic  centimetres  this  mallein  does 
not  cause  an  elevation  of  temperature  exceeding  0.5°  to  0.8°.  But 
one  cubic  centimetre  injected  into  a  horse  having  glanders  causes  its 
temperature  to  mount  to  40°  C.,  and  at  the  point  of  inoculation  a  con- 
siderable swelling  is  developed  which  lasts  from  four  to  six  days — 
in  healthy  horses  a  swelling  the  size  of  a  man's  fist  is  developed  at 
the  point  of  inoculation,  which  disappears  within  twenty -four  hours. 
In  Pasteur's  laboratory,  according  to  Nocard  (1892),  mallein  is 
prepared  as  follows :  The  glanders  bacillus  is  first  made  so  virulent 
by  successive  inoculations  in  susceptible  animals  that  it  will  kill  a 
rabbit  or  a  white  mouse  in  a  few  hours.  This  virulent  bacillus  is  cul- 
tivated in  glycerin-peptone-flesh-infusion  (five  per  cent  of  glycerin  and 
five  per  cent  of  peptone).  The  cultures  are  kept  in  the  incubating 
oven  for  four  weeks  at  a  temperature  of  31°  C.,  and  then  sterilized  in 
the  autoclave  at  110°  C.  They  are  then  filtered  through  paper  and 
evaporated,  in  vacuo,  over  sulphuric  acid,  at  a  low  temperature,  to  one- 
tenth  of  the  original  volume.  The  result  is  a  syrup-like,  dark-brown, 
strong-smelling  liquid,  which  is  about  one-half  glycerin.  This  can 
be  preserved  in  a  cool  and  dark  place  for  a  long  time.  When  it  is  to 
be  used  nine  parts  of  a  0.5-per-cent  solution  of  carbolic  acid  are 
added  to  one  part  of  the  glycerin  extract.  The  concentrated  extract, 
when  injected  into  a  healthy  horse  in  the  dose  of  one-half  to  one  cubic 
centimetre,  causes  a  local  swelling  which  disappears  after  two  or  three 
days.  The  temperature  of  the  body  is  elevated  from  1.5°  to  2°  C.  as 
a  result  of  the  injection,  and  there  are  chilliness,  loss  of  appetite,  and 
debility.  When  the  diluted  mallein  is  injected  in  healthy  animals  in 


320  PROTECTIVE    INOCULATIONS. 

the  dose  of  2.5  cubic  centimetres  no  reaction  occurs.  On  the  other 
hand,  this  dose  causes  an  intense  febrile  reaction  in  horses  with  glan- 
ders. There  is  a  chill  followed  by  an  elevation  of  temperature  amount- 
ing to  2°  to  3°  C.,  accompanied  by  dyspnoea  and  great  debility;  in 
some  cases  the  animal  dies  as  a  result  of  the  inoculation. 

For  the  preparation  of  the  active  substance  in  a  dry  condition, 
Foth  gives  the  following  directions :  The  cultures  are  evaporated  at 
a  temperature  not  exceeding  80°  C.  to  one-tenth  of  their  volume,  and 
filtered.  The  clear  and  thick,  'dark-brown  liquid  is  then  slowly 
dropped  into  absolute  alcohol  (twenty-five  to  thirty  parts)  with  con- 
stant stirring.  A  flaky,  white  precipitate  is  thrown  down,  and  ac- 
cumulates as  a  pale  yellow  mass  upon  the  sides  and  bottom  of  the 
vessel.  After  standing  for  twenty-four  hours  the  alcohol  is  carefully 
drawn  off  and  the  precipitate  washed  with  absolute  alcohol.  This 
is  to  be  carefully  done,  and  to  avoid  loss  will  require  several  days. 
The  precipitate  is  then  placed  upon  a  thick  paper  filter  and  thor- 
oughly washed  by  drawing  alcohol  through  it  by  means  of  an  exhaus- 
tion apparatus,  after  which  the  purified  precipitate  is  collected  and 
dried  with  care  at  a  low  temperature — best  in  a  vacuum  over  sul- 
phuric acid.  A  spongy,  crumbling  mass  is  thus  obtained,  which  is 
easily  crushed  to  form  an  extremely  light  white  powder.  This  is 
readily  soluble  in  water.  It  is  not  at  all  hygroscopic,  and  can  be 
preserved  in  a  dry  condition  without  difficulty .  The  dose  for  a  horse 
is  0.1  gramme. 

De  Schweinitz  and  Kilborne,  in  a  paper  published  in  November, 
1892,  state  that  in  December,  1890,  they 

"extracted  from  culture  liquids  of  the  Bacillus  malleus  an  albumose  which 
appeared  to  be  the  active  principle  in  these  cultures.  At  that  time  a  pre- 
liminary experiment  was  conducted  to  see  if  this  substance  could  be  used 
to  make  guinea-pigs  immune  to  the  disease — glanders.  The  result  was  that 
out  of  a  set  of  five,  three  vaccinated  and  two  checks,  only  one,  a  vaccinated 
animal,  recovered  from  an  inoculation  of  a  glanders  culture.  This  experi- 
ment has  since  been  repeated  with  sets  of  ten  and  twelve  guinea-pigs  each, 
with,  at  present  writing,  only  negative  results.  A  note  of  this  work  was 
published  in  the  '  Annual  Report  of  the  Department  of  Agriculture  for  1891.' 
The  albumose  was  best  obtained  from  the  cultures,  after  the  removal  of  the 
germ,  by  means  of  a  Pasteur  filter,  by  precipitation  with  absolute  alcohol, 
resolution  in  water,  and  reprecipitation." 

Babes  (1892)  claims  to  have  succeeded  in  immunizing  guinea-pigs 
against  glanders  by  means  of  the  toxic  substances  contained  in  cul- 
tures of  the  bacillus. 

Foth  (1894)  has  reported  the  results  of  extended  experiments 
which  have  been  made  with  his  "  Malleinum  siccum  "  in  Austro-Hun- 
gary.  These  results  are  stated  as  follows : 


PROTECTIVE    INOCULATIONS.  321 

The  experiments  were  for  the  most  part  made  by  Professor  Schin- 
delka,  of  Vienna.  The  tests  were  made  with  doses  ranging  from  0.1 
gramme  to  0.2  gramme.  The  number  of  horses  treated,  for  diagnos- 
tic purposes,  was  four  hundred  and  fifty-five;  of  these  one  hundred 
and  forty-seven  were  examined  post  mortem.  In  general  the  infected 
horses  reacted  and  the  others  did  not.  A  reaction  of  2C  C.  and  up- 
ward, running  a  typical  course,  was  evidence  that  the  animal  was  in- 
fected, and  such  animals  were  killed  and  carefully  examined  by  au- 
topsy. 

A  reaction  of  1.3°  to  1.9°  C.,  running  a  typical  course,  was  taken 
as  evidence  that  the  animal  was  probably  infected,  and  called  for  its 
isolation  and  a  subsequent  inoculation  after  an  interval  of  four  weeks. 

A  reaction  of  less  than  1.2°  C.,  or  an  atypical  course  of  the  febrile 
reaction,  was  taken  as  evidence  of  non-infection. 

The  typical  febrile  reaction  consisted  in  a  rapid  or  gradual  eleva- 
tion, according  to  the  dose,  then  a  fall  of  some  tenths  of  a  degree,  a 
subsequent  elevation  to  the  highest  previously  reached  point  or  above, 
and  a  gradual  fall  to  the  normal.  The  atypical  reaction,  which  some- 
times occurs  in  healthy  animals,  consists  in  an  early  and  rapid  eleva- 
tion followed  by  an  equally  rapid  fall  to  the  normal.  To  properly 
distinguish  the  typical  temperature  curve,  upon  which  the  diagnosis 
depends,  hourly  observations  are  considered  necessary. 

Schiitz  (1894),  as  a  result  of  his  experiments  on  fifty-four  horses, 
arrives  at  the  conclusion  that  malleiu  may  give  rise  to  the  so-called 
"  typical  reaction  "  in  horses  which  are  not  infected  with  glanders. 

Hutyra  and  Preiz  (1894),  as  a  result  of  their  extended  researches, 
arrive  at  the  conclusion  that  the  use  of  malleiu  constitutes  the  most 
important  means  for  the  early  diagnosis  of  glanders  in  horses.  They 
conclude  that  a  temperature  of  39.4°  C.  may  be  accepted  as  a  safe 
positive  mallein  reaction.  According  to  them  the  reaction  commences 
from  four  to  six  hours  after  the  injection,  and  reaches  its  maximum 
in  from  eight  to  fourteen  hours — rarely  in  sixteen  to  twenty  hours. 
The  return  to  the  normal  occurs  in  from  twenty -four  to  thirty-six 
hours.  The  authors  last  named  give  the  following  directions  for  the 
preparation  of  mallein :  The  virulence  of  the  glanders  bacillus  is  first 
increased  by  passing  it  through  a  series  of  guinea-pigs.  Cultures 
are  then  made  upon  sterilized  potato.  When  the  culture  and  potato 
have  become  quite  dry  and  dark  colored  they  are  collected  in  a  glass 
dish  and  covered  with  a  liquid  consisting  of  equal  parts  of  glycerin 
and  distilled  water,  containing  three  to  five  parts  per  thousand  of 
mercuric  chloride.  After  standing  for  from  ten  to  fourteen  days  in 
an  incubating  oven  at  37.5°  C.,  the  liquid. is  filtered  through  paper 
21 


322  PROTECTIVE   INOCULATIONS. 

and  sterilized  for  an  hour  in  a  steam  sterilizer.  This  liquid  remains 
sterile  on  account  of  the  presence  of  mercuric  chloride,  and  may  be 
preserved  a  long  time  without  losing  its  activity.  The  dose  is  from 
0.3  to  0.5  cubic  centimetre,  which  is  diluted  to  three  cubic  centimetres 
with  carbolic  acid  water  (0.5-per-cent  solution).  The  diluted  solu- 
tion may  also  be  kept  a  long  time  without  losing  its  activity. 

Bonome  and  Vivaldi  (1892)  have  tested  the  action  of  mallein  ob- 
tained by  precipitation  with  alcohol  upon  various  animals.  Guinea- 
pigs  were  found  to  resist  comparatively  large  doses  (ten  to  fifteen 
milligrammes),  while  rabbits  and  cats  were  more  sensitive  to  the 
toxic  action.  In  guinea-pigs  and  rabbits  infected  with  glanders  ba- 
cilli very  small  doses  had  a  favorable  influence  upon  the  progress  of 
the  infection,  and  in  healthy  guinea-pigs  a  certain  degree  of  immunity 
was  induced  by  the  repeated  injection  of  small  doses. 

In  a  subsequent  paper  (1894)  Bonome  reports  that  he  has  had 
favorable  results  in  the  treatment  of  chronic  glanders  in  man  by  doses 
of  yV  to  ^¥  cubic  centimetre.  The  first  dose  is  said  to  have  caused 
an  elevation  of  temperature,  headache,  polyuria,  etc.,  but  upon  re- 
peating the  dose  after  two  or  three  days  a  decided  improvement  of 
the  general  symptoms  followed. 

Chenot  and  Picq  (1892)  claim  to  have  cured  glanders  in  guinea-pigs 
by  injections  of  blood  serum  from  the  ox,  which  animal  has  an  im- 
munity from  the  disease.  They  also  state  that  the  blood  serum  of 
the  ox  is  germicidal  for  the  glanders  bacillus.  Guinea-pigs  treated 
with  ox  serum,  either  before  or  after  infection,  recovered  in  seven 
cases  out  of  ten.  When  inoculated  with  very  virulent  cultures,  which 
usually  killed  these  animals  in  five  days,  the  animals  are  said  to  have 
survived  from  twenty-one  to  forty-two  days. 

Bonome  (1894)  reports  his  success  in  curing  infected  guinea-pigs 
by  means  of  filtered  cultures  made  in  the  blood  serum  of  the  ox.  He 
was  not,  however,  successful  in  accomplishing  this  result  with  mallein 
made  in  the  usual  way. 

HOG   CHOLERA. 

The  experiments  thus  far  made  with  reference  to  protective  inocu- 
lations against  hog  cholera  have  not  given  very  satisfactory  results. 
Selander  and  Metchnikoff  have  reported  success  in  immunizing  rab- 
bits, but  according  to  Smith  their  experiments  were  made  with  the 
bacillus  of  swine  plague,  and  not  with  that  of  hog  cholera  as  they 
supposed.  The  following  conclusions  have  been  formulated  by  Smith 
as  a  result  of  his  extended  experiments : 

"1.  It  is  possible  to  produce  immunity  toward  hog-cholera  and  swine- 


PROTECTIVE   INOCULATIONS.  323 

plague  bacteria  in  the  very  susceptible  rabbit  and  the  less  susceptible  guinea- 
pig.  In  the  rabbit  the  only  promising-  method  of  immunization  toward  hog 
cholera  is  the  use  of  gradually  augmented  doses  of  attenuated  cultures. 

"2.  Immunization  toward  swine  plague  is  produced  artificially  with 
much  greater  ease  than  toward  hog-cholera  bacteria. 

"3.  The  blood  serum  of  animals  protected  against  hog  cholera  and  swine 
plague  is  almost  as  efficacious  in  producing  immunity  soon  after  treatment 
as  the  bacterial  products  obtained  from  cultures. 

"4.  Different  degrees  of  culture  in  both  hog  cholera  and  swine  plague 
lead  to  different  forms  of  the  inoculation  disease.  The  greater  the  immunity 
short  of  complete  protection  the  more  prolonged  and  chronic  the  disease  in- 
duced subsequently  by  inoculation. 

"5.  Pathogenic  bacteria  may  remain  in  the  organs  of  inoculated  animals 
some  time  after  apparently  full  recovery.  Their  presence  may  or  may  not 
be  associated  with  lesions  recognizable  by  the  naked  eye. 

"  6.  The  toxicity  of  sterilized  cultures  appears  to  be  directly  proportional 
to  the  number  of  bacteria  in  the  injected  fluid." 

The  experiments  of  Moore,  reported  in  Bulletin  No.  6  of  the 
Bureau  of  Animal  Industry,  show  that  the  bacillus  of  hog  cholera 
does  not  become  attenuated  by  being  passed  through  rabbits,  and  that 
in  the  experiments  of  Metclmikoff,  which  led  him  to  conclude  that 
this  is  the  case,  the  bacillus  of  swine  plague,  and  not  that  of  cholera, 
was  used. 

De  Schweinitz  studied  the  chemical  products  of  the  hog-cholera 
bacillus  in  1890,  and  obtained  from  the  cultures  cadaverin,  methyl- 
amine,  a  ptomaine  ("sucholotoxin"),  and  an  albumose  ("sucholoal- 
bumin  "). 

Novy  (1890)  has  also  obtained,  by  Brieger's  method,  a  basic  toxic 
substance  ("susotoxin")  which  kills  rats  in  the  dose  of  0.125  to  0.25 
cubic  centimetre.  He  also  obtained  from  concentrated  cultures,  by 
precipitation  with  absolute  alcohol,  a  toxalbumin  which,  when  dried, 
killed  rats  in  three  or  four  hours  in  the  dose  of  0.05  to  0.01  gramme. 

De  Schweinitz  in  a  later  publication  (1899)  reports  that  he  has 
obtained,  by  the  method  of  Brieger  and  Boer  for  the  isolation  of  the 
diphtheria  antitoxin,  an  ash-free  white  powder,  which  possesses  the 
antitoxic  properties  of  serum  from  an  immune  animal;  ninety  cubic 
centimetres  of  serum  gave  him  0.152  gramme  of  this  powder.  The 
method  referred  to  consists  in  precipitation  by  the  use  of  zinc  sul- 
phate, repeated  solution  in  sodium  hydrate  and  precipitation  by 
CO,.  In  preparing  serum  for  his  experiments,  cattle,  horses,  mules, 
and  monkeys  were  employed.  "  The  animals  received  injections 
of  the  filtered,  sterile  or  live,  cultures  of  the  hog-cholera  germ  and 
swine-plague  germ,  respectively,  or  the  solutions  of  their  products, 
including  cell  contents,  extracts,  and  secretions.  These  injections 
were  made  either  subcutaneously,  intravenously,  or  intra-abdomi- 
nally,  or  a  combination  of  two  or  more  of  these  methods,  depending 


324  PROTECTIVE    INOCULATIONS. 

upon  the  results  obtained.  The  quantities  given  at  first  were  small, 
but  increased  gradually  until  large  amounts  of  the  material  used  could 
be  injected  without  bad  results.  This  treatment  of  the  animals  must 
be  carried  out  very  carefully,  and  requires  six  to  eight  months'  time 
before  the  serum  is  sufficiently  potent  to  be  of  any  practical  use.  As 
the  treatment  continues,  the  power  of  the  serum  to  check  the  motility 
of  the  hog-cholera  germ  increases  with  rapidity.  The  length  of  im- 
munity produced  by  the  injection  of  serum  is  short,  and  more  perma- 
nent immunity  can  apparently  be  secured  by  using  in  addition  to 
serum  the  products  of  the  germs." 

The  results  of  extensive  inoculations  (thirty-five  thousand  animals) 
which  have  been  made  by  the  Agricultural  Department  during  the  past 
two  years  have  not  yet  been  published,  but  it  is  understood  that  as  a 
rule  these  results  have  been  quite  satisfactory. 

HOG   ERYSIPELAS. 

Pasteur's  first  studies  relating  to  the  etiology  of  "rouget"  were 
made,  in  collaboration  with  Chamberland,  Roux,  and  Thuillier,  in 
1882.  Pasteur  found  that  the  virulence  of  his  cultures  was  increased 
by  passing  them  through  pigeons  and  diminished  by  passing  them 
through  rabbits.  By  a  series  of  inoculations  in  rabbits  he  obtained 
an  attenuated  virus  suitable  for  protective  inoculations  in  swine.  In 
practice  he  recommended  the  use  of  a  mild  virus  first,  and  after  an 
interval  of  twelve  days  of  a  stronger  virus.  These  inoculations  have 
been  extensively  practised  in  France,  and  the  fact  that  immunity  may 
be  established  in  this  way  is  well  demonstrated.  There  has  been  some 
doubt,  however,  as  to  the  practical  value  of  the  method,  as  its  appli- 
cation has  been  attended  with  some  loss,  and  there  appears  to  be 
danger  that  the  disease  may  be  spread  by  the  alvine  discharges  of 
inoculated  animals.  In  a  region  where  the  annual  losses  from  the 
disease  are  considerable,  and  where  the  soil  is,  perhaps,  thoroughly 
infected  with  the  bacilli,  protective  inoculations  probably  afford  the 
best  security  against  loss.  But  when  it  is  practicable  to  stamp  out 
the  disease  by  quarantine  of  infected  animals,  disinfection  of  localities 
in  which  cases  have  occurred,  and  strict  attention  to  cleanliness,  this 
will  probably  be  found  the  best  method  of  combating  the  malady. 

Chamberland  (1894)  states  that  in  the  preceding  seven  years,  dur- 
ing which  time  protective  inoculations  were  practised  in  France  on  a 
large  scale,  the  mortality  from  rouget  has  been  reduced  to  1.45  per 
cent,  whereas  before  these  inoculations  were  practised  the  mortality 
from  this  disease  was  about  twenty  per  cent.  Losses  amounting  in 


PROTECTIVE    INOCULATIONS.  325 

some  instances  to  as  much  as  ten  per  cent  have  resulted  from  the  in- 
oculations. These  are  ascribed  by  Chambeiiand  to  secondary  infec- 
tion, through  the  inoculation  wound,  with  other  pathogenic  bacteria. 

Jakobi  (1888)  reports  the  results  of  inoculations  made  in  1887  and 
1888  with  "vaccines"  obtained  from  Pasteur's  agent  in  Paris.  His 
results  agree  with  those  previously  reported  by  Lydtin  in  showing  a 
smaller  loss,  as  &  result  of  the  inoculations,  among  young  pigs  than 
among  older  ones — over  sixteen  weeks.  The  loss  among  young  pigs 
was  only  1.3  per  cent.  The  animals  which  survived  subsequently  es- 
caped infection,  while  others  not  inoculated,  associated  with  them, 
succumbed  to  the  disease. 

Hutyra  has  given  the  following  statistics  of  inoculations  made  in 
Hungary  during  the  year  1889,  with  "  vaccines  "  obtained  from  the 
Pasteur  laboratory  in  Vienna:  48,637  pigs  were  inoculated  on  117 
different  farms.  Of  these  143  (0.29  per  cent)  died  between  the  first 
and  second  inoculation.  After  the  second  inoculation  59  animals  died 
(0.1  per  cent).  During  the  year  following  the  inoculations,  1,082  in- 
oculated pigs  died  of  Rothlauf.  Before  the  inoculations  the  annual 
loss  in  the  same  localities  is  said  to  have  been  from  10  to  30  per 
cent.  Upon  one  farm  220  pigs  which  had  been  inoculated  were  as- 
sociated with  1,500  not  inoculated.  The  loss  among  the  latter  was 
50  per  cent;  among  the  former  2.27  per  cent. 

In  a  later  communication  (1894)  Jakobi  gives  the  following  results 
of  inoculations  made  since  by  the  same  method:  1889,  inoculated 
133,  loss  5;  1890,  inoculated  151,  loss  2;  1891,  inoculated  158,  loss 
0;  1893,  inoculated  223,  loss  0;  1894,  inoculated  145,  loss  4.  Total 
inoculated,  1,036;  total  loss,  14.  These  inoculations  were  made  upon 
19  different  farms,  and  principally  upon  pigs  less  than  four  months 
old.  The  inoculated  pigs  were  isolated  to  prevent  the  communication 
of  the  disease  to  other  unprotected  pigs. 

Inoculations  with  Blood  Serum  of  Immune  Animals. — The  experi- 
ments of  Lorenz,  commenced  in  1891,  seem  to  establish  the  fact  that 
there  is  an  antitoxin  in  the  blood  of  animals  which  have  an  acquired 
immunity  against  this  disease  which  may  be  used  for  producing  im- 
munity in  other  animals,  or  for  the  cure  of  the  disease  in  animals 
already  infected.  In  his  latest  communication  (1894)  Lorenz  says : 

% '  When  I  read  in  the  journals  of  the  discovery  of  Behring  and  Kitasato 
that  the  blood  of  animals  immunized  against  tetanus,  when  injected  beneath 
the  skin  of  other  animals,  gave  them  an  immunity  against  tetanus,  I  had  in 
my  possession  rabbits  which  were  immunized  against  Rothlauf.  I  took  from 
one  of  these  some  blood  from  the  ear  vein,  injected  it  under  the  skin  of  a 
mouse,,  inoculated  this  latter  with  a  Rothlauf  culture,  and  made  the  dis- 
covery, in  this  and  a  series  of  subsequent  experiments,  that  the  blood  of  an 


326  PROTECTIVE   INOCULATIONS. 

animal  immune  against  Rothlauf  contains  an  immunizing  substance.  I 
further  ascertained  that  this  substance  is  found  only  in  the  blood  serum,  and 
not  in  the  solid  portions  of  the  body  organs,  etc.,  and  with  the  exception  of 
the  blood  was  found  only  in  the  secretions  of  serous  membranes.  I  also 
found  that  the  immunizing  substance  is  only  to  be  found  for  a  certain  time 
after  renewed  infection  in  the  immune  animals,  and  that  it  gradually  disap- 
pears, without  the  loss  of  immunity  in  the  animal,  however.  Finally,  I  dis- 
covered that  the  animals  into  which  one  injects  blood  serum  from  immune 
animals  do  not  acquire  a  lasting  immunity,  but  are  only  immune  for  a  rela- 
tively short  time/' 

In  experiments  made  in  1893  and  1894,  with  a  view  to  producing 
immunizing  serum  for  protective  inoculations  on  a  large  scale,  Lorenz 
met  with  some  disappointments ;  but  he  proposes  to  renew  his  attempts 
and  hopes  to  avoid  the  difficulties  which  have  been  brought  to  light 
by  experience,  one  of  which  he  states  as  follows : 

"When  an  animal  already  immunized  against  Rothlauf  receives  an  in- 
jection of  a  considerable  quantity  of  a  culture  of  the  bacillus,  in  order  to 
cause  the  production  in  its  blood  of  a  serum  of  high  therapeutic  value,  the 
animal  bears  these  injections  without  any  notable  reaction.  But  its  blood 
serum  contains  during  the  following  days,  besides  the  immunizing  substance, 
also  poisonous  substances,  and  blood  which  is  taken  too  soon  (twenty-four 
hours)  after  the  injection  has  a  toxic  action  upon  animals  which  are  already 
infected.  If  this  poisonous  serum  is  injected  into  a  mouse  which  has  been 
infected  two  days  before  with  Rothlauf  bacilli,  in  the  dose  of  about  0.05  cubic 
centimetre,  death  occurs  in  a  few  hours,  even  when  scarcely  any  evidence  of 
sickness  had  been  observed  before  the  injection.'' 

The  fact  that  mice  infected  with  this  bacillus  may  be  cured  by  in- 
jecting into  them  blood  serum  from  an  immunized  rabbit  has  also 
been  demonstrated  by  F.  Klemperer  (1892) .  In  his  experiments  with 
the  bacillus  of  mouse  septicaemia,  and  with  Friedlander's  bacillus,  he 
found  that  serum  from  an  immune  rabbit  may  be  used  to  immunize 
mice  and  also  to  cure  them  after  infection,  while  serum  from  a  non- 
immune  rabbit  has  no  such  action.  The  immunity  produced  in  this 
way  was  found  to  be  specific.  That  is,  animals  immunized  against 
the  pathogenic  action  of  one  of  these  bacilli  were  not  protected  against 
infection  by  the  other.  The  "  Heilsemm  "  when  added  to  cultures  in 
vitro  did  not  prove  to  have  any  special  bactericidal  action. 

HYDROPHOBIA. 

Notwithstanding  the  extended  researches  made,  especially  in  Pas- 
teur's laboratory,  the  etiology  of  hydrophobia  still  remains  unsettled. 
It  has  been  demonstrated  by  experiment  that  the  virus  of  the  disease 
is  located  in  the  brain,  spinal  marrow,  and  nerves  of  animals  which 
have  succumbed  to  the  disease,  as  well  as  in  the  salivary  secretions 
of  rabid  animals ;  and  that  the  disease  may  be  transmitted  by  intra- 
venous inoculation,  or  by  introducing  a  small  quantity  of  virus  beneath 


PROTECTIVE   INOCULATIONS. 

the  dura  mater,  with  greater  certainty  than  by  subcutaneous  inocula- 
tions. But  the  exact  nature  of  this  virus  has  not  been  determined. 
The  fact  that  a  considerable  interval  elapses  after  inoculation  before 
the  first  symptoms  are  developed  indicates  that  there  is  a  multiplica- 
tion of  the  virus  in  the  body  of  the  infected  animal ;  and  this  is  further 
shown  by  the  fact  that  after  death  the  entire  brain  and  spinal  marrow 
of  the  animal  have  a  virulence  equal  to  that  of  the  material  with  which 
it  was  inoculated  in  the  first  instance.  The  writer's  experiments 
(1887)  show  that  this  virulence  is  neutralized  by  a  temperature  of  60° 
C.,  maintained  for  ten  minutes— a  temperature  which  is  fatal  to  all 
known  pathogenic  bacteria  in  the  absence  of  spores.  But  recent  ex- 
periments show  that  certain  toxic  products  of  bacterial  growth  are 
destroyed  by  the  same  temperature.  We  are,  therefore,  not  justified 
in  assuming  that  the  morbid  phenomena  are  directly  due  to  the  pres- 
ence of  a  living  micro-organism ;  and,  indeed,  it  seems  probable,  from 
what  we  already  know,  that  the  symptoms  developed  and  the  death  of 
the  animal  are  due  to  the  action  of  a  potent  chemical  poison  of  the 
class  known  as  toxalbumins.  But,  if  this  is  true,  we  have  still  to  ac- 
count for  the  production  of  the  toxic  albuminoid  substance,  and,  in 
the  present  state  of  knowledge,  have  no  other  way  to  explain  its  in- 
crease in  the  body  of  the  infected  animal  than  the  supposition  that  a 
specific,  living  germ  is  present  in  the  virulent  material,  the  introduc- 
tion of  which  into  the  body  of  a  susceptible  animal  gives  rise  to  the 
morbid  phenomena  characterizing  an  attack  of  rabies. 

Pasteur  and  his  associates  have  thus  far  failed  to  demonstrate  the 
presence  of  microorganisms  in  the  virulent  tissues  of  animals  which 
have  succumbed  to  an  attack  of  rabies.  Babes  has  obtained  micro- 
cocci  in  cultures  from  the  brain  and  spinal  cord  of  rabid  animals,  and 
states  in  his  article  on  hydrophobia  in  "Les  Bacteries  "  (second  edi- 
tion, p.  791)  that  pure  cultures  of  the  second  and  third  generation  in- 
duced rabies  in  susceptible  animals ;  but  his  own  later  researches  do 
not  appear  to  have  established  the  etiological  relation  of  this  micro- 
coccus. 

Gibier  (1884)  has  reported  the  presence  of  spherical  refractive 
granules,  resembling  micrococci,  in  the  brain  of  rabid  animals,  which 
he  demonstrated  by  rubbing  up  a  little  of  the  cerebral  substance  with 
distilled  water.  As  these  supposed  micrococci  did  not  stain  with 
the  usual  aniline  colors  and  were  not  cultivated,  it  appears  very  doubt- 
ful whether  the  refractive  granules  seen  were  really  microorganisms. 

Fol  (18S5)  claims  to  have  demonstrated  the  presence  of  minute 
cocci,  0.2  fj.  in  diameter,  in  sections  of  spinal  cord  from  rabid  ani- 
mals, by  Weigert's  method  of  staining.  The  cords  were  hardened  in 


328  PROTECTIVE    INOCULATIONS. 

a  solution  of  bichromate  of  potash  and  sulphate  of  copper,  colored 
with  a  solution  of  hrematoxylon,  and  decolorized  in  a  solution  of  fer- 
rocyanide  of  potash  and  borax. 

The  writer  (1887)  has  made  similar  preparations,  carefully  follow- 
ing the  method  as  described  by  Fol,  but  was  not  able  to  demonstrate 
the  presence  of  microorganisms  in  the  numerous  sections  made.  Nor 
have  the  observations  of  Fol  been  confirmed  by  the  researches  of  other 
bacteriologists  who  have  given  their  attention  to  the  subject  since  the 
publication  of  his  paper. 

Pasteur  first  announced  his  success  in  reproducing  rabies  in  sus- 
ceptible animals  by  inoculations  of  material  "  from  the  medulla  oblon- 
gata,  the  frontal  lobes  of  the  cerebral  hemispheres,  and  the  cerebro- 
spinal  fluid  "  in  a  communication  to  the  Academy  of  Sciences  made  on 
May  30th,  1881.  At  the  same  time  he  reported  his  success  in  the 
discovery  of  "  a  method  for  considerably  shortening  the  period  of  in- 
cubation in  rabies,  and  also  of  reproducing  the  disease  with  certainty." 
This  was  by  inoculations  made  after  trephining,  upon  the  surface  of 
the  brain  with  material  obtained  from  the  brain  of  a  rabid  animal. 
Dogs  inoculated  in  this  way  developed  rabies  in  the  course  of  two 
weeks,  and  died  before  the  end  of  the  third  week — sometimes  of  furi- 
ous rabies  and  sometimes  of  the  paralytic  form  of  the  disease.  In  a 
second  communication  (December  llth,  1882)  Pasteur  reports  his 
success  in  communicating  the  disease  by  the  intravenous  injection  of 
virus  from  the  central  nervous  system ;  also  the  experimental  demon- 
stration of  the  fact  that  all  forms  of  rabies  may  be  produced  by  the 
same  virus ;  also  that  all  portions  of  the  spinal  cord  of  rabid  animals 
are  virulent,  as  well  as  all  parts  of  the  brain ;  also  that  an  animal 
(dog)  which  had  recovered  from  a  mild  attack  after  inoculation  proved 
to  be  subsequently  immune,  and  that  "  this  observation  constitutes  a 
first  step  toward  the  discovery  of  the  prophylaxis  of  rabies."  On 
February  25th,  1884,  many  important  facts  are  stated  which  had  been 
developed  during  the  continuous  study  of  the  disease,  and  among 
others  the  fact  that  by  passing  the  virus  through  a  series  of  animals 
of  the  same  species  a  fixed  degree  of  virulence  is  established  for  each 
susceptible  species,  as  shown  by  a  definite  and  uniform  period  of  in- 
cubation. By  this  method  a  virus  had  been  obtained  which  produced 
rabies  in  rabbits  in  seven  or  eight  days,  and  another  which  caused 
the  development  of  the  disease  in  guinea-pigs  in  five  or  six  days  after 
inoculation.  In  a  subsequent  communication  (May  19th,  1884)  evi- 
dence is  given  to  show  that  by  successive  inoculations  in  monkeys  the 
period  of  incubation  is  prolonged,  and  that  the  attenuated  virus  ob- 
tained from  a  monkey,  after  several  successive  inoculations  in  this 


PROTECTIVE   INOCULATIONS. 

animal,  when  inoculated  into  the  dog,  no  longer  produces  fatal  rabies ; 
and  that  dogs  so  treated  are  subsequently  immune. 

In  his  address  before  the  International  Medical  Congress  at  Copen- 
hagen (August  llth,  1884),  after  a  review  of  the  facts  developed  during 
his  experimental  researches  made  during  the  preceding  four  years, 
Pasteur  gives  an  account  of  the  test  made  by  a  commission,  appointed 
by  the  Minister  of  Public  Instruction,  to  determine  the  efficacy  of  his 
method  as  applied  to  the  protection  of  dogs.  He  says  that  he  gave  to 
the  commission  nineteen  dogs  which  had  been  rendered  refractory 
against  rabies  by  preventive  inoculations.  These  nineteen  dogs  and 
nineteen  control  animals,  obtained  from  the  pound  without  any  selec- 
tion, were  tested  at  the  same  time.  The  test  was  made  upon  some  of 
the  animals  of  both  series  by  inoculation  with  virulent  material  upon 
the  surface  of  the  brain,  and  upon  others  by  allowing  them  to  be  bit- 
ten by  rabid  dogs,  and  upon  still  others  by  intravenous  inoculations. 

Not  one  of  the  protected  animals  developed  rabies ;  on  the  other 
hand,  three  of  the  control  dogs  out  of  six  bitten  by  a  mad  dog  devel- 
oped the  disease,  five  out  of  seven  which  received  intravenous  inocu- 
lations died  of  rabies,  and  five  which  were  trephined  and  inoculated 
on  the  surface  of  the  brain  died  of  the  same  disease.  In  a  subsequent 
report  the  commission,  of  which  M.  Boulley  was  president,  stated  that 
twenty-three  protected  dogs  which  were  bitten  by  ordinary  mad  dogs 
all  remained  in  perfect  health,  while  sixty-six  per  cent  of  the  control 
animals,  bitten  in  the  same  way,  developed  rabies  within  two  months. 

In  his  communication  of  October  26th,  1885,  Pasteur  reports  his 
discovery  of  the  fact  that  the  virulence  of  the  spinal  cord  of  a  rabbit 
is  gradually  attenuated  by  hanging  it  in  a  dry  atmosphere,  and  is 
finally  entirely  lost ;  also  that  he  had  been  able  to  make  a  practical 
application  of  this  discovery  in  the  protection  of  dogs  by  means  of 
successive  inoculations  beneath  the  skin  of  an  emulsion  of  spinal  mar- 
row attenuated  in  this  way.  The  first  inoculation  was  to  be  made 
with  a  portion  of  spinal  cord  which  had  been  kept  long  enough  to  de- 
prive it  of  all  virulence,  and  this  was  followed  by  daily  inoculations 
with  more  virulent  material,  until  finally  material  was  used  from  a 
cord  only  a  day  or  two  old. 

With  reference  to  his  first  inoculations  in  man,  Pasteur  says : 

"  Making-  use  of  this  method,  I  have  already  made  fifty  dogs  of  various 
races  and  ages  immune  to  rabies,  and  -had  not  met  with  a  single  failure, 
when,  on  the  6th  of  July,  quite  unexpectedly,  three  persons,  residents  of 
Alsace,  presented  themselves  at  my  laboratory." 

These  persons  were  Theodore  Yone,  who  had  been  bitten  on  the 
arm  on  July  4th ;  Joseph  Meister,  aged  nine,  bitten  on  the  same  day 


330  PROTECTIVE   INOCULATIONS. 

by  the  same  rabid  dog ;  and  the  mother  of  Meister,  who  had  not  been 
bitten.  The  child  had  been  thrown  down  by  the  dog  and  bitten  upon 
the  hand,  the  legs,  and  the  thighs,  in  all  in  fourteen  different  places. 
Pasteur  commenced  the  treatment  on  July  6th,  by  injecting  beneath 
the  skin  of  this  child  an  emulsion  of  cord  which  had  been  kept  for 
fourteen  days;  this  was  followed  by  twelve  more  inoculations  made 
on  successive  days  with  cord  of  increasing  degrees  of  virulence — the 
last  with  cord  a  day  old.  On  March  1st,  1886,  Pasteur  reported  to 
the  Academy  of  Sciences  the  fact  that  the  boy  Meister  remained  in 
good  health  and  gave  detailed  information  with  reference  to  a  number 
of  cases  which  had  since  been  treated  by  the  same  method. 

With  reference  to  the  duration  of  the  immunity  resulting  from 
these  inoculations  Pasteur  says  (1886)  that  out  of  fourteen  dogs  in- 
oculated with  "ordinary  street  virus,"  by  trephining,  at  the  expiration 
of  a  year  after  the  protective  inoculations  had  been  practised,  eleven 
resisted ;  out  of  six  tested  in  the  same  way  at  the  end  of  two  years 
two  proved  to  be  immune. 

In  November,  1886,  Pasteur  communicated  to  the  Academy  of 
Sciences  the  results  of  his  experiments  with  reference  to  a  modification 
of  his  method  as  at  first  employed — the  so-called  intensive  method. 
This  modification  consisted  in  making  the  inoculations  with  cords  of 
increasing  virulence  in  more  rapid  succession. 

The  method  followed  at  Odessa,  as  reported  by  Gamaleia  (1887), 
is  shown  below,  the  day  being  given  above  and  age  of  the  cord 
below. 

1  2          _3^        45         6         7  8910 

14-13      12-11      10-9      8-7      6-5      4-3      2-10      8-642 

Since  the  adoption  of  this  method  and  the  use  of  larger  quantities 
of  virus,  according  to  Gamaleia,  there  have  been  no  deaths  among 
those  inoculated,  numbering  more  than  two  hundred  at  the  time  the 
report  was  made.  The  author  last  referred  to  concludes  from  his  ex- 
perience that  "  the  mortality  diminishes  in  direct  relation  to  the  quan- 
tity of  the  vaccine  injected." 

Bujwid  (1889)  reports  a  total  of  670  inoculations,  with  9  deaths, 
made  at  Varsovie  during  the  years  1886, 1887,  and  1888.  His  method 
is  shown  below. 

1  2  34567 

12-10         8-643643' 

The  results  of  inoculations  made  at  the  Pasteur  Institute  in  Paris 
during  the  years  1886  to  1890  are  given  in  the  following  table : 


PROTECTIVE   INOCULATIONS. 


331 


Year. 

Number  Treated. 

Died. 

Mortality. 

1886    

2,671 

25 

0  94 

1887  

1,770 

13 

0.73 

1888  

1,622 

9 

0.55 

1889  

1,830 

6 

0.33 

1890      

1.540 

5 

0.32 

Total  

9,433 

58 

0.61 

In  the  following  table,  A  includes  all  persons  treated  who  had  been 
bitten  by  an  animal  proved  to  be  rabid ;  B,  persons  bitten  by  animals 
examined  by  veterinary  surgeons  and  pronounced  rabid;  C,  persons 
bitten  by  animals  suspected  of  being  rabid.  The  figures  relate  to  the 
year  1890: 


Number  Treated. 

Died. 

Mortality. 

A     

416 

0 

B  

909 

4 

0.44 

C  

215 

1 

0.46 

Bordoni-Uffreduzzi  gives  the  following  statistics  with  reference  to 
the  inoculations  practised  at  the  Pasteur  Institute  in  Turin  during 
the  years  1886  to  1891 :  81  persons  were  inoculated  by  the  method 
first  proposed  by  Pasteur,  with  a  mortality  of  2.46  per  cent;  925 
persons  were  subsequently  inoculated  by  the  same  method,  but  with 
larger  doses  of  virus,  with  a  mortality  of  1.72  per  cent.  Finally,  338 
persons  were  inoculated  with  still  larger  doses,  with  a  mortality  of 
0.29  per  cent. 

At  the  Pasteur  Institute  in  Palermo  the  number  of  persons  inocu- 
lated in  the  four  years  prior  to  1891  was  662,  with  a  mortality  among 
the  inoculated  of  0.6  per  cent.  In  Bologna  (1890)  210  persons  bitten 
by  dogs  undoubtedly  mad  were  inoculated,  with  a  mortality  of  0.47 
per  cent. 

In  the  Pasteur  Institute  at  Naples  810  persons  were  treated  during 
the  years  1886  to  1892,  with  a  mortality  of  0.86  per  cent. 

During  the  year  1891,  1,564  persons  were  inoculated  at  the  Pas- 
teur Institute  in  Paris,  with  a  total  mortality  of  0.57  per  cent.  In 
324  of  these  cases  the  animal  which  inflicted  the  bite  was  proved  to 
be  rabid  by  experimental  inoculations. 

Horsely  (1889)  has  made  a  comparison  of  the  results  obtained  by 
the  "  intensive  treatment "  as  compared  with  these  by  the  treatment 
first  employed,  and  says : 


PROTECTIVE   INOCULATIONS. 

"  It  is  evident  that  the  intensive  treatment  is  very  successful  in  coping 
with  the  worst  cases,  and  that,  instead  of  being-  itself  a  source  of  death,  as 
asserted  by  those  who  gain  notoriety  and  subsistence  by  villifying  and  mis- 
representing scientific  progress,  it  is  a  powerful  agent  in  saving  life." 

The  following  table  is  given  by  Horsely  "  as  showing  the  contrast 
between  the  old  or  simple  treatment  and  the  intensive  treatment " : 

Simple  Treatment,  1886.  Intensive  Treatment,  1888. 

Odessa 3.39  per  cent.  0.64  per  cent. 

Warsaw 4.1  0.0*      " 

Moscow 8.2 f      "  1.6 

*  The  figures  include  sixteen  months'  work,  and  thirty  individuals  bitten  in  the 
face — four  by  wolves. 

f  This  unusually  high  rate  was  found  to  be  due  to  imperfections  in  the  manner 
of  preparing  the  cords  for  the  inoculation  material. 

Perdrix  (1890),  in  an  analysis  of  the  results  obtained  at  the  Pasteur 
Institute  in  Paris,  calls  attention  to  the  fact  that  the  mortality  among 
those  treated  has  diminished  each  year  and  ascribes  this  to  improve- 
ment in  the  method.  He  says : 

' '  At  the  outset  it  was  difficult  to  know  what  formula  to  adopt  for  the 
treatment  of  each  particular  case.  Upon  consulting  the  accounts  of  the  bites 
in  persons  who  have  died  of  hydrophobia  notwithstanding  the  inoculations, 
we  have  arrived  at  a  more  precise  determination  as  to  the  treatment  suitable 
for  each  case,  according  to  the  gravity  of  the  lesions.  In  the  cases  with  seri- 
ous wounds  we  inject  larger  quantities  of  the  emulsion  of  cord  and  repeat  the 
inoculations  with  the  most  virulent  material.  For  the  bites  upon  the  head, 
which  are  especially  dangerous,  however  slight  their  apparent  gravity  may 
be,  the  treatment  is  more  rapid,  and,  above  all,  more  intensive — that  is  to 
say,  the  virulent  cord  is  injected  several  times." 

The  statistics  arranged  with  reference  to  the  location  of  the  bite 
are  given  by  Perdrix  as  follows : 

Bitten  upon  the  head,        684;  died,  12  =  1.75  per  cent. 
".  hands,  4,396;      "        9  =  0.2 
"     limbs,    2,839;      "        5  =  0.17 

Other  methods  of  making  susceptible  animals  immune  against 
hydrophobia  have  been  proposed  and  proved  by  experiment  to  be 
successful.  Thus  Galtier  in  1880-1881  claimed  that  the  sheep  and 
the  goat  could  be  protected  by  intravenous  injections  of  the  virus  of 
rabies,  and  more  recent  experiments  fully  confirm  this.  Protopopoff 
(1888)  by  injecting  an  emulsion  of  cord  from  a  rabid  animal  into  the 
circulation  of  dogs  succeeded  in  protecting  them  from  hydrophobia 
as  a  result  of  subsequent  inoculation  with  virulent  material  upon  the 
surface  of  the  brain.  He  injected  into  a  vein,  at  intervals  of  three 
days,  one  cubic  centimetre  of  an  emulsion  of  cord — first  of  six  days, 
second  of  three  days,  third  of  one  day.  Roux  had  previously  accom- 
plished the  same  result  by  a  single  intravenous  injection  of  a  larger 


PROTECTIVE    INOCULATIONS.  333 

quantity  (thirty -five  cubic  centimetres)  of  cord  which  had  been  kept 
for  five  or  six  days.  In  discussing  his  results  Roux  calls  attention  to 
the  fact,  which  had  been  developed  during  his  experiments,  that  the 
virulence  of  the  spinal  cord  of  rabid  animals  does  not  depend  entirely 
upon  the  length  of  time  it  has  been  kept,  but  that  large  doses  of  cord 
kept  as  long  as  twelve  days  will  sometimes  produce  hydrophobia  when 
injected  into  the  circulation  of  dogs,  when  smaller  doses  of  cord  kept 
five  or  six  days  prove  to  be  inoffensive.  He  supposes  that  during 
desiccation  the  virus  may  not  be  equally  acted  upon  throughout  the 
cord,  but  that  certain  "  islands  "  in  the  central  portion  may  remain 
living  and  virulent  when  all  the  rest  has  been  modified.  A  practical 
point  with  reference  to  the  preservation  of  virulent  material  is  referred 
to  by  Roux  in  a,  note  published  in  the  Annals  of  the  Pasteur  Insti- 
tute. This  is  the  fact  that  when  preserved  in  glycerin,  portions  of 
the  central  nervous  system  retain  their  virulence  for  considerable 
time.  Other  forms  of  virus,  e.g.,  vaccine,  may  also  be  preserved  in 
the  same  way. 

Centanni  (1892)  has  succeeded  in  making  rabbits  immune  by  in- 
oculating them  with  an  attenuated  virus  obtained  by  subjecting  viru- 
lent material  to  the  action  of  an  artificial  gastric  juice.  After  digestion 
for  less  than  twelve  hours  the  virus  still  kills  rabbits,  when  inoculated 
beneath  the  dura  mater,  but  the  period  of  incubation  is  considerably 
prolonged.  After  from  twelve  to  twenty  hours'  digestion  it  no  longer 
kills  rabbits,  but  causes  an  infection,  from  which  they  recover,  and 
after  which  they  are  immune. 

Serum-therapy. — Tizzoni  and  Centanni  (1892)  have  reported  suc- 
cess in  the  treatment  of  infected  rabbits  by  the  use  of  blood  serum 
from  immune  animals  of  the  same  species — immunized  by  the  "  Ital- 
ian method  "  above  described.  The  animals  experimented  upon  were 
inoculated  with  a  "street  virus"  which  produced  paralytic  rabies  in 
rabbits  and  caused  their  death  in  from  fourteen  to  eighteen  days. 
The  blood  serum  was  obtained  from  rabbits  which  had  been  proved 
to  be  immune  by  resisting  inoculations  of  virus  of  full  strength  on 
the  surface  of  the  brain.  The  blood  serum,  in  doses  of  three  to  five 
cubic  centimetres,  was  injected  subcutaneously,  or  into  the  peritoneal 
cavity,  or  into  the  circulation.  Injections  were  made  into  each  animal 
(in  all  from  eleven  to  twenty-six  cubic  centimetres)  after  the  first  symp- 
toms of  paralytic  rabies  had  appeared  (on  the  seventh,  the  tenth,  the 
eleventh,  and  the  fourteenth  day  after  infection) .  Four  rabbits  treated 
in  this  way  fully  recovered.  In  a  subsequent  experiment  the  bacteri- 
ologists named  treated  three  rabbits  with  a  dry  antitoxin  obtained  by 
precipitation  from  the  blood  serum  of  immune  rabbits.  The  precipi- 


334  PROTECTIVE   INOCULATIONS. 

tate  was  obtained  by  adding  one  part  of  serum  to  ten  parts  of  alcohol, 
and  was  dried  in  vacuo.  This  dried  precipitate,  in  doses  of  0.18  to 
0.25  gramme,  was  dissolved  in  sterilized  water  and  injected  as  in  the 
previous  experiment.  Commencing  on  the  eighth  day  after  infection 
five  or  six  doses  were  given— in  all  0.9  to  1.3  gramme.  All  of  the  ani- 
mals treated  recovered,  while  all  of  the  control  animals  died.  Babes 
had  previously  (1889)  reported  successful  results  in  conferring  im- 
munity upon  susceptible  animals  by  injections  of  blood  serum  from 
immune  animals. 

Tizzoni  and  Schwartz,  in  pursuing  this  line  of  investigation  (1892), 
report  that  while  the  blood  serum  of  immune  rabbits  neutralizes  the 
"fixed  virus"  of  rabies  in  vitro,  after  short  contact  (five  hours),  the 
blood  serum  of  immune  dogs  has  but  slight  antitoxic  potency.  The 
immunizing  substance  in  the  rabbit  serum  does  not  dialyze,  is  soluble 
in  glycerin,  is  precipitated  by  alcohol,  and  in  general  behaves  like  a 
globulin.  In  subsequent  experiments  Tizzoni  and  Schwartz  used 
blood  serum  from  dogs  and  rabbits  immunized  by  Pasteur's  method. 
The  blood  was  drawn  from  the  carotid  of  the  immune  animals,  and 
the  serum  from  the  same,  mixed  with  virulent  spinal  marrow  in  the 
form  of  a  homogeneous  emulsion,  obtained  by  crushing  and  pressing 
through  linen.  These  experiments  corresponded  with  those  pre- 
viously made  as  to  the  superior  antitoxic  power  of  rabbit  serum, 
which,  after  five  hours'  contact,  neutralized  the  virulence  of  the  emul- 
sion of  cord.  By  the  injection  of  serum  from  an  immune  rabbit,  in 
doses  of  five  cubic  centimetres,  into  the  circulation  of  other  rabbits, 
they  were,  as  a  rule,  made  immune.  The  immunizing  substance 
(antitoxin)  was  shown  by  other  experiments  to  be  present  only  in  the 
blood.  Extracts  from  the  liver,  spleen,  kidney,  or  muscles  gave  a 
negative  result. 

In  a  later  communication  (1894)  Tizzoni  and  Centanni  give  an 
account  of  further  experiments  made  principally  upon  sheep  and  dogs. 
By  repeated  inoculations  they  succeeded  in  obtaining  from  these  ani- 
mals a  serum  having  an  immunizing  value  of  1 : 25, 000  or  more,  and 
from  this  a  precipitate  was  obtained  estimated  to  have  a  value  of 
1:300,000,  and  which  in  doses  of  0.23  gramme  (of  the  dried  precipi- 
tate), dissolved  in  five  times  its  weight  of  water,  ought  to  be  a  sufficient 
dose  to  protect  a  man  from  the  development  of  hydrophobia  after 
being  bitten  by  a  rabid  animal. 

The  authors  named  believe  that  inoculations  with  this  antitoxin 
would  be  reliable  for  man,  and  that  they  would  possess  decided  ad- 
vantages over  Pasteur's  method  of  inoculation.  These  advantages  are 
specified  as  follows : 


PROTECTIVE   INOCULATIONS.  335 

"  Applicability  at  any  time  during1  the  period  of  incubation  up  to  the 
moment  of  the  appearance  of  symptoms  of  rabies  ;  absolute  absence  of  viru- 
lence and  of  any  injurious  action  :  very  rapid  treatment  by  the  injection  of 
one  or  several  small  doses  of  material :  complete  solubility  and  consequently 
prompt  absorption  of  the  material  injected  and  its  easy  preservation  in  a  dry 
condition.*' 

INFLUENZA. 

The  bacillus  discovered  by  Pfeiffer,  in  1892,  is  now  well  estab- 
lished as  the  specific  cause  of  this  disease.  Bruschettini  has  recently 
(1893)  reported  the  details  of  his  experiments  upon  rabbits,  for  which 
animals  this  bacillus  is  pathogenic.  As  a  result  of  these  experiments 
he  has  reached  the  following  conclusions : 

"  1.  Eabbits  may  be  vaccinated  against  the  pathogenic  action  of  cultures 
of  the  influenza  bacillus  without  great  difficulty. 

"2.  The  best  material  for  producing  a  high  grade  of  immunity  is  blood 
cultures  which  have  been  filtered  through  the  Berkenfeld  filter. 

"3.  The  blood  serum  of  immunized  animals  has  strong  antitoxic  proper- 
ties, but  has  no  germicidal  power. 

"4.  The  serum  of  vaccinated  animals  has  the  power  of  conferring  im- 
munity upon  other  animals,  in  comparatively  small  amounts — in  the  pro- 
portion of  1 : 42,000  of  body  weight,  and  perhaps  still  less. 

"5.  This  serum  has  also  a  decided  curative  action,  and  rescues  rabbits 
from  death  even  as  late  as  forty-eight  hours  after  infection  by  injection  of  a 
culture  of  the  bacillus  into  the  trachea." 

These  results  lead  the  author  to  hope  that  serum-therapy  may 
afford  a  method  of  curing  this  disease  in  man.  For  this  purpose  the 
blood  of  an  immune  rabbit  would  appear  to  be  the  most  promising 
source  from  which  to  procure  an  antitoxic  serum. 

INFLUENZA   IN    HORSES. 

SCHUTZ  (1887)  has  described  a  minute  oval  bacillus,  usually  asso- 
ciated in  pairs,  which  appears  to  be  the  specific  infectious  agent  in 
the  disease  known  in  Germany  as  Brusteeuche.  This  bacillus  is  path- 
ogenic for  mice,  rabbits,  pigeons,  and  guinea-pigs,  but  not  for  swine 
or  chickens.  By  injection  of  cultures  into  the  parenchyma  of  the 
lungs  Schiitz  reproduced  the  disease — confirmed  in  1888  by  Hell. 

Horses  which  have  suffered  an  attack  of  infectious  influenza  are 
subsequently  immune,  and  the  experiments  of  Hell  have  shown  that 
an  immunity  also  follows  the  disease  which  results  from  inoculations 
with  pure  cultures  of  the  Schiitz  bacillus. 

The  extended  experiments  made  by  the  War  Department  of  the 
German  Government  show  that  the  disease  is  not  produced  by  intra- 
venous injections  or  by  the  ingestion  of  the  bacillus  with  the  food. 
Infection  occurs,  however,  when  cultures  are  injected  into  the  re- 


336  PROTECTIVE   INOCULATIONS. 

spiratory  passages.  Subcutaneous  injections  cause  a  painful  local 
tumefaction,  often  followed  by  an  abscess,  but  without  the  general 
symptoms  of  influenza. 

Experiments  have  been  made  in  Germany  by  Hell,  Siedamgrotzki, 
and  others,  which  indicate  that  the  subcutaneous  injection  of  blood 
serum  from  immune  horses  may  confer  immunity  on  other  horses. 
Hell  usually  injected  forty  cubic  centimetres  at  a  time,  and  repeated 
this  at  intervals  until  two  hundred  to  two  hundred  and  forty  cubic 
centimetres  had  been  injected  in  the  course  of  two  or  three  weeks. 
He  also  reports  the  results  of  treatment  by  injections  of  blood  serum 
into  the  trachea  in  horses  already  infected,  and  thinks  these  injections 
had  a  favorable  influence  on  the  course  of  the  disease.  Experiments 
made  subsequently  by  Toepper  have  given  a  similar  result,  but  others 
have  not  been  so  fortunate,  and  the  immunizing  value  of  blood-serum 
injections,  as  practised  by  the  authors  referred  to,  seems  to  be  still  a 
matter  of  some  doubt.  Toepper  (1893)  gives  full  directions  for  col- 
lecting the  serum  and  a  detailed  account  of  results  of  experimental 
inoculations  made  by  himself  and  others.  He  prefers  to  inject  the 
serum  into  the  breast  over  the  ensiform  cartilage.  No  reaction  oc- 
curs after  the  injection. 

PLEURO-PNEUMONIA    OF   CATTLE. 

Protective  inoculations  against  this  disease  have  long  been  success- 
fully practised.  For  this  purpose  serum  obtained  from  the  lungs  of 
an  animal  recently  dead  has  been  employed,  this  having  been  proved 
by  experiment  to  be  infectious  material,  although  the  exact  nature  of 
the  infectious  agent  present  in  it  was  not  determined. 

Willems,  who  wras  one  of  the  first  to  advocate  the  use  of  protective 
inoculations  in  pleuro-pneumonia  (1852),  gave  a  lecture  in  1894  in 
which  he  reviewed  the  evidence  in  favor  of  these  inoculations  in 
the  disease  under  consideration.  Various  methods  had  been  em- 
ployed. Thus  Willems  states  that  the  natives  of  the  banks  of  the 
Zambeze  cause  animals  to  swallow  a  certain  quantity  of  the  liquid 
from  the  pleural  cavity  of  an  animal  recently  dead,  and  thus  give 
them  immunity.  The  virus  has  been  injected  into  the  circulation  by 
some  experimenters,  and  others  have  )roposed  to  attenuate  it  by  heat. 
But  the  method  which  has  been  most  extensively  employed  is  that 
discovered  by  the  Dutch  settlers  at  the  Cape  of  Good  Hope  (the 
Boers),  and  consists  in  inoculating  animals  in  the  tail  with  serum 
from  the  lungs  of  an  animal  recently  dead ;  or  with  a  virus  obtained 
from  the  tumefaction  produced  by  such  an  inoculation  in  the  tail. 


PROTECTIVE    INOCULATIONS.  337 

This  secondary  virus  was  very  extensively  used  by  Lenglen,  a  veter- 
inarian at  Arras,  who  communicated  his  results  to  the  Academy  of 
Science  at  Paris,  in  April,  1863,  and  Willems  says,  in  his  last  pub- 
lished communication,  that  this  is  the  method  which  he  prefers.  It 
is  also  the  method  most  extensively  employed  in  Australia,  into  which 
country  infectious  pleuro-pneumonia  was  introduced  in  1858.  It 
(juickly  spread  and  has  caused  enormous  losses.  The  killing  of  all 
animals,  sick  or  suspected  of  being  infected,  was  tried  for  several 
years;  but  this  proved  to  be  ineffectual  for  stamping  out  the  disease, 
and  the  sacrifice  was  so  great  that  this  measure  of  prophylaxis  was 
abandoned. 

According  to  Loir,  attention  in  Australia  was  called  to  Willems' 
method  of  protective  inoculations,  in  1861,  by  a  letter  from  Cape 
Colony  published  in  the  journals  of  Sydney  and  in  Melbourne.  The 
method  was  at  once  applied  both  in  Victoria  and  in  New  South  Wales, 
and  since  that  date  many  thousands  of  cattle  have  been  inoculated. 
In  order  to  obtain  a  sufficient  supply  of  virus  the  method  recom- 
mended by  Pasteur  in  1882  has  been  followed.  This  is  described  by 
Pasteur  himself  in  the  following  words : 

"With  a  single  lung  we  may  procure  sufficient  virus  to  serve  for  numer- 
ous series  of  animals.  And  without  having  recourse  to  other  lungs  this  pro- 
vision may  be  maintained  in  the  following  manner :  It  is  sufficient  before  the 
supply  of  virus  is  exhausted  to  inoculate  a  young  calf  in  the  dewlap  or  in 
the  shoulder.  The  animal  dies  very  promptly,  and  all  its  tissues  near  the 
point  of  inoculation  are  infiltrated  with  serum,  which  is  virulent,  and  may  be 
collected  and  preserved  in  a  state  of  purity." 

Loir  prefers  to  obtain  the  virus  in  this  way  from  a  calf  six  to  twelve 
months  old,  during  the  second  week  after  inoculation,  when  the  tem- 
perature of  the  animal  has  gone  up  to  40°  to  42°  C.,  as  the  virus  is 
then  said  to  possess  the  maximum  degree  of  intensity.  This  vaccine 
seems  to  become  attenuated  in  passing  through  a  series  of  animals  by 
inoculation,  so  that  when  it  has  been  passed  through  a  series  of  five 
animals  it  no  longer  produces  death  even  when  inoculated  in  the  most 
dangerous  localities.  Loir  testifies  to  the  protective  value  of  inocula- 
tions with  this  virus  made  in  the  tail  of  the  animal,  and  gives  the  fol- 
lowing example :  A  few  months  prior  to  the  publication  of  his  paper 
(1893),  about  two  thousand  cows  were  inoculated  with  a  virus  which 
had  been  passed  through  a  series  of  five  calves.  At  the  moment  of 
being  driven  away  they  were  joined  by  nineteen  other  cows  not  vac- 
cinated. After  being  on  the  road  for  a  distance  of  two  thousand  kilo- 
metres, the  animals  arrived  at  their  destination.  The  two  thousand 
vaccinated  were  in  good  condition,  while  eight  of  the  non-vaccinated 

had  died  of  pleuro-pneumouia. 
22 


338  PROTECTIVE   INOCULATIONS. 

In  the  Bulletin  of  the  Central  Society  of  Veterinary  Medicine  of  May 
24th,  1894,  M.  Kobcis  reports  the  results  of  inoculations  made  with 
cultures  of  Arloing's  Pneumobacillus  liquefaciens  bovis,  and  with  in- 
jections of  pulmonary  serum.  His  statistics  with  reference  to  the 
last-mentioned  "legal"  inoculations  he  has  obtained  from  official 
documents  relating  to  the  Department  of  the  Seine. 

The  total  number  of  infected  localities  in  this  department  during 
the  years  1885  to  1891  was  1,253;  total  number  of  contaminated  ani- 
mals, 18,356 ;  total  number  inoculated,  18,359 ;  total  number  of  deaths 
prior  to  inoculation,  1,753;  total  number  of  deaths  after  inoculation, 
2,741;  total  number  of  deaths  due  to  the  inoculation,  94;  total  per- 
centage of  mortality,  22.8  per  cent.  After  discussing  these  and  other 
statistics  Robcis  arrives  at  the  conclusion  that  Arloing's  method  of 
preventive  inoculations  with  cultures  of  the  Pneumobacillus  liquefaciens 
bovis  gives  better  results  than  the  legal  method  with  serum  from  an 
infected  animal,  the  total  loss  among  animals  exposed  to  contagion 
not  being  over  twelve  to  fourteen  per  cent. 

Nocard  (1892)  says  that  serum  from  the  lungs  of  an  animal  dead 
from  pleuro-pneumonia  preserves  its  virulence  and  usefulness  as  a 
vaccine,  when  mixed  with  half  a  volume  of  pure  neutral  glycerin  and 
half  a  volume  of  a  five-per-cent  solution  of  carbolic  acid.  At  the  end 
of  two  and  a  half  months  this  mixture  preserved  its  full  virulence. 

PNEUMONIA. 

The  micrococQus  of  croupous  pneumonia  was  discovered  by  the 
present  writer  in  the  blood  of  rabbits  inoculated  subcutaneously  with 
his  own  saliva  in  September,  1880.  In  1885  this  micrococcus,  which 
I  had  repeatedly  obtained  in  pure  cultures  from  the  blood  of  rabbits 
inoculated,  as  in  the  first  instance,  with  my  own  saliva,'  was  identified 
with  the  micrococcus  of  the  same  form  present  in  the  rusty  sputum 
of  patients  with  pneumonia.  In  a  paper  read  before  the  Pathological 
Society  of  Philadelphia,  in  April,  1885,  and  published  in  the  Ameri- 
can Journal  of  Medical  Sciences  on  July  1st  of  the  same  year,  I  say : 

"It  seems  probable  that  this  micrococcus  is  concerned  in  the  etiology  of 
croupous  pneumonia,  and  that  the  infectious  nature  of  the  disease  is  due  to 
its  presence  in  the  fibrinous  exudate  into  the  pulmonary  alveoli." 

This  has  since  been  fully  established  by  the  researches  of  Frankel, 
Weichselbaum,  Netter,  Gameleia,  and  many  others.  Frankel  first 
discovered  this  micrococcus  in  his  own  salivary  secretions  in  1883, 
and  his  first  paper  relating  to  its  presence  in  the  exudate  of  croupous 
pneumonia  was  published  on  July  13th,  1885,  i.e.,  thirteen  days  after 


PROTECTIVE   INOCULATIONS.  339 

the  publication  of  tlie  paper  from  which  the  above  quotation  is  made. 
Under  these  circumstances  the  writer  feels  justified  in  again  calling 
attention  to  his  priority  in  the  discovery  of  this  important  pathogenic 
niicrococcus,  and  in  objecting  to  its  beiug  described  as  "Frankel's 
pneumococcus, "  the  "  diplococcus  of  Frankel,"  etc. 

In  my  paper  above  referred  to  (July,  1885)  I  described  this  micro- 
coccus  under  the  name  of  Micrococcus  Pasteuri,  but  in  my  "  Manual 
of  Bacteriology  "  (1892)  it  is  described  under  the  name  of  Micrococcus 
pneumonias  crouposce. 

This  micrococcus  is  very  pathogenic  for  mice  and  for  rabbits,  less 
so  for  guinea-pigs  and  for  dogs.  Like  other  pathogenic  microorgan- 
isms of  the  same  class,  it  varies  greatly  in  virulence  when  obtained 
from  different  sources.  In  the  saliva  of  healthy  persons,  which 
seems  to  be  its  normal  habitat,  it  sometimes  has  comparatively  little 
virulence.  On  the  other  hand,  when  contained  in  the  blood  or  in  an 
exudate  from  a  serous  cavity  of  an  infected  rabbit  or  mouse,  it  is  very 
virulent.  In  one  instance  (1881)  the  writer  has  seen  a  fatal  result  in 
a  dog  from  the  subcutaneous  injection  of  one  cubic  centimetre  of  bloody 
serum  from  the  subcutaneous  connective  tissue  of  a  rabbit  recently 
dead. 

Pneumonia  never  results  from  subcutaneous  injections  into  sus- 
ceptible animals,  but  injections  through  the  thoracic  walls  into  the 
lung  may  induce  a  typical  fibrinous  pneumonia.  This  was  first  de- 
monstrate,d  by  Talamon  (1883),  who  injected  the  fibrinous  exudate  of 
croupous  pneumonia,  obtained  after  death,  or  drawn  during  life  by 
means  of  a  Pravaz  syringe,  from  the  hepatized  portion  of  the  lung, 
into  the  lungs  of  rabbits.  Gameleia  has  also  induced  pneumonia  in 
a  large  number  of  rabbits,  and  also  in  dogs  and  sheep,  by  injections 
directly  into  the  pulmonary  tissue.  Sheep  were  found  to  survive  sub- 
cutaneous inoculations,  unless  very  large  doses  (five  cubic  centimetres) 
of  a  virulent  culture  were  injected.  But  intrapulinonarj'  inoculations 
are  said  to  have  invariably  produced  a  typical  fibrinous  pneumonia 
which  usually  proved  fatal.  In  dogs  similar  injections  gave  rise  to 
a  "frank,  fibrinous  pneumonia  which  rarely  proved  fatal,  recovery 
usually  occurring  in  from  ten  to  fifteen  days,  after  the  animal  had 
passed  through  the  stages  of  red  and  gray  hepatization  characteristic 
of  this  affection  in  man." 

Without  doubt  an  attack  of  pneumonia  is  followed  by  a  certain 
degree  of  immunity  of  longer  or  shorter  duration.  According  to 
Huge,  who  has  made  a  careful  study  of  the  subject,  relapses  are 
very  infrequent — indicating  a  temporary  immunity — but  subsequent 
attacks  are  more  likely  to  occur  in  those  who  have  once  suffered  an 


340  PROTECTIVE  INOCULATIONS. 

attack  of  the  disease,  and  as  many  as  four  or  five  attacks  Lave  been 
known  to  occur  in  the  same  individual. 

In  1,100  cases  collected  by  Wagner  but  2  relapses  occurred 
(=0.18  per  cent).  Huge  reports  that  in  440  cases  treated  at  the 
Oharite  in  Berlin  there  were  but  2  relapses.  The  liability  to  sub- 
sec  juent  attacks  at  a  later  period  is  shown  by  the  following  figures, 
which  we  copy  from  Euge's  paper:  In  280  cases  reported  by  Stortz, 
26.4  per  cent  had  previously  suffered  an  attack  of  the  disease;  in 
133  cases  reported  by  Morhart  the  proportion  of  previous  attacks  was 
41.3  per  cent;  in  157  cases  by  Pohlmann,  34.4  per  cent;  in  166  cases 
by  Schapira,  31.3  per  cent;  in  128  cases  by  Keller,  36.9  per  cent;  in 
175  cases  by  Grisolle,  30.9  per  cent. 

The  writer,  in  a  series  of  experiments  made  during  the  winter  of 
1880-81,  obtained  experimental  evidence  which  showed  that  suscep- 
tible animals  (rabbits)  acquire  immunity  from  the  pathogenic  action 
of  this  micrococcus  as  a  result  of  inoculations  with  an  attenuated 
virus.  The  experiments  referred  to  had  as  their  object  the  determi- 
nation of  the  comparative  value  of  various  germicidal  agents,  as  tested 
upon  this  micrococcus ;  incidentally  it  was  found  "  that  a  protective 
influence  has  been  shown  to  result  from  the  injection  "  (into  rabbits) 
"  of  virus,  the  virulence  of  which  has  been  modified,  without  being 
entirely  destroyed,  by  the  agent  used  as  a  disinfectant."  (Quoted 
from  the  writer's  report  of  the  experiments  referred  to,  "Studies 
from  Biological  Laboratory,"  Johns  Hopkins  University,  Balti- 
more, 1882.) 

In  1891  G.  and  F.  Klemperer  published  an  important  memoir  re- 
lating to  the  pathogenic  action  of  this  micrococcus  and  the  production 
of  immunity  in  susceptible  animals  by  means  of  filtered  cultures.  In 
some  cases  this  immunity  was  found  to  last  as  long  as  six  months.  A 
curious  fact  developed  in  their  researches  was  that  the  potency  of  the 
substance  contained  in  the  filtered  cultures  was  increased  by  subject- 
ing these  to  a  temperature  of  41°  to  42°  C.  for  three  or  four  days,  or 
to  a  higher  temperature  (60°  C. )  for  an  hour  or  two.  When  injected 
into  a  vein  after  being  subjected  to  such  a  temperature,  immunity  was 
complete  at  the  end  of  three  or  four  days ;  but  the  same  material,  not 
so  heated,  required  larger  doses  and  a  considerably  longer  time  (four- 
teen days)  to  confer  immunity  upon  a  susceptible  animal.  The  un- 
warmed  material  caused  a  considerable  elevation  of  temperature,  last- 
ing for  some  days.  The  authors  mentioned  conclude  from  their 
investigations  that  the  toxic  substance  present  in  cultures  of  Micro- 
coccus  pneumoniae  crouposse  is  a  proteid  substance,  which  they  propose 
to  call  pneumotoxin.  The  substance  produced  in  the  body  of  an  im- 


PROTECTIVE    INOCULATIONS.  341 

mune  animal,  as  a  result  of  protective  inoculations,  upon  which  the 
immunity  of  these  animals  depends,  is  also  a  proteid,  which  they  call 
antipneurnotoxin.  This  they  isolated  from  the  blood  serum  of  im- 
mune animals.  By  experiment  they  were  able  to  demonstrate  that 
the  blood  serum  containing  this  protective  proteid,  when  injected 
into  other  animals,  rendered  them  immune;  and  also  that  it  arrested 
the  progress  of  the  infectious  malady  induced  by  inoculating  suscep- 
tible animals  with  virulent  cultures  of  the  micrococcus.  When  in- 
jected into  the  circulation  of  an  infected  animal,  its  curative  action 
was  manifested  by  a  considerable  reduction  of  the  body  temperature. 
The  toxalbumin  was  obtained  from  filtered  bouillon  cultures  of  a  viru- 
lent variety  of  the  micrococcus  of  pneumonia,  in  the  form  of  an  amor- 
phous, yellowish-white  powder.  This  was  thrown  down  from  the 
filtered  cultures  by  means  of  alcohol,  and  again  dissolved  in  water 
and  reprecipitated  in  order  to  purify  it. 

Issaeff  (1893)  as  a  result  of  his  experiments  has  found  that  the 
virulence  of  this  micrococcus  can  be  greatly  increased  by  successive 
inoculations  in  the  peritoneal  cavity  of  rabbits,  and  that  after  a  series 
of  ten  or  twelve  such  inoculations  the  blood  of  the  infected  animal  does 
not  coagulate  and  becomes  extremely  toxic.  In  order  to  obtain  the 
toxins  from  this  blood,  Issaeff  collects  the  blood  of  three  or  four  ani- 
mals just  dead  in  a  sterilized  vessel,  and  adds  to  this  an  equal  volume 
of  sterilized  water  containing  one  per  cent  of  glycerin,  made  alkaline 
by  the  addition  of  a  few  drops  of  a  concentrated  solution  of  bicarbo- 
nate of  soda.  The  mixture  is  sterilized  by  passing  it  through  a  Cham- 
berland  filter.  This  liquid  sometimes  kills  rabbits  when  injected  into 
the  circulation  in  the  proportion  of  one  per  cent  of  the  weight  of  the 
animal.  When  heated  to  70°  C.  its  toxic  power  is  considerably  di- 
minished, and  a  temperature  of  100°  C.  neutralizes  it  completely. 

Emmerich  (1891)  has  succeeded  in  immunizing  rabbits  and  mice 
by  the  intravenous  injection  of  a  very  much  diluted  but  virulent  cul- 
ture of  the  micrococcus.  Other  rabbits  and  mice  were  rendered  im- 
mune by  injecting  into  them  material  obtained  from  rabbits  immu- 
nized with  diluted  cultures.  The  flesh  of  these  animals  was  rubbed  up 
into  a  pulp,  and  the  juices  were  obtained  by  pressure  through  a  piece 
of  sterilized  cloth.  The  bloody  juice,  after  standing  for  twelve  hours 
at  a  temperature  of  10°  C.,  was  passed  through  a  Pasteur  filter  and 
then  served  to  immunize  the  animals  referred  to. 

Belfanti  (1892)  has  succeeded  in  immunizing  rabbits  against  the 
pathogenic  action  of  this  micrococcus  by  injecting  into  the  circulation 
a  filtrate  obtained  from  the  sputa  of  pneumonia  cases.  The  viscid 
sputa  mixed  with  an  equal  part  of  distilled  water  was  kept  on  ice  for 


PROTECTIVE   INOCULATIONS. 

twenty -four  Lours  and  then  passed  through  a  Chamber  land  filter. 
Ten  cubic  centimetres  of  this  filtrate  was  injected  into  the  ear  vein  of 
rabbits.  Some  of  the  animals  so  treated  proved  to  be  immune  against 
general  infection  when  inoculated  with  a  virulent  culture  of  the  micro- 
coccus,  but  they  had  a  localized  inflammation  and  oedema  about  the 
point  of  inoculation.  After  recovering  from  this  they  proved  to  be 
entirely  refractory  against  subsequent  inoculations. 

Foa  and  Scabia  (1892)  have  reported  success  in  producing  immu- 
nity with  filtered  cultures,  and  also  with  a  glycerin  extract  from  the 
blood  of  an  infected  rabbit.  This,  after  filtration,  was  injected  sub- 
cutaneously  in  doses  of  two  cubic  centimetres  at  intervals  of  five  days. 
The  authors  named  have  also  produced  immunity  in  rabbits  by  the 
use  of  "  pneumo-protein. "  This  is  an  extract  from  the  bacterial  cells 
obtained  by  first  collecting  these  from  the  surface  of  a  Chamberland 
filter  through  which  the  cultures  have  been  passed;  then  digesting 
them  for  three  hours  at  55°  C.  in  a  five-per-cent  solution  of  glycerin. 
According  to  Foa  and  Scabia  immunity  produced  in  this  way  is  more 
decided  and  of  longer  duration  than  that  resulting  from  the  other 
methods  tested  by  them. 

Mosny  (1892)  has  also  made  numerous  experiments  which  show 
that  rabbits  may  be  immunized  by  means  of  filtered  cultures,  or  by 
the  juices  from  the  tissues  of  an  immune  animal  obtained  by  macer- 
ation and  filtration.  When  sterilized  cultures  were  employed  the  best 
results  were  obtained  by  first  heating  very  virulent  cultures  for  three 
hours  at  60°  C.  The  dose  employed  was  ten  cubic  centimetres,  and 
immunity  was  not  established  immediately  but  required  a  period  of 
at  least  four  days  for  its  development. 

The  blood  serum  of  immune  rabbits  was  not  found  to  have  any 
bactericidal  power,  and  the  micrococcus  of  pneumonia  preserved  its 
vitality  longer  in  the  blood  serum  of  immune  rabbits  than  in  that  of 
other  animals  of  the  same  species. 

G.  and  F.  Klemperer  had  previously  reported  that  the  blood  of 
immune  rabbits  does  not  destroy  the  micrococcus  of  pneumonia  or  re- 
strict its  development. 

Issaeff  (1893)  also  reports  his  success  in  immunizing  rabbits  by 
means  of  sterilized  cultures  or  filtered  blood  from  infected  animals 
recently  dead.  A  single  intravenous  injection  of  ten  cubic  centi- 
metres of  filtered  blood,  prepared  as  heretofore  indicated  (p.  340), 
sufficed  to  confer  immunity.  To  test  immunity  the  animals  were 
subsequently  inoculated  with  two  to  four  drops  of  virulent  blood ;  and 
to  maintain  it  the  inoculations  (0.5  cubic  centimetre)  were  repeated 
every  four  weeks.  Although  immune  against  infection  these  animals 


PROTECTIVE   INOCULATIONS.  o43 

are  said  not  to  have  acquired  any  immunity  against  the  toxins  of  the 
micrococcus  of  pneumonia.  Contrary  to  the  conclusion  reached  by 
G.  and  F.  Klemperer,  Issaeff  concludes  from  his  experiments  that 
"rabbits,  although  completely  refractory  against  pneumonic  infec- 
tion, remain  highly  sensitive  to  the  toxins  of  this  microbe.  Even 
small  doses  of  the  toxins  are  not  neutralized  in  the  blood  of  vacci- 
nated animals.  We  are  therefore  brought  to  the  conclusion  that  the 
existence  of  an  antitoxic  property  of  the  blood  of  vaccinated  animals 
cannot  be  admitted." 

The  serum  of  immunized  rabbits  was  not  found  by  Issaeff  to  pos- 
sess any  bactericidal  power  for  the  micrococcus  of  pneumonia,  and 
no  attenuation  of  virulence  occurred  as  a  result  of  cultivation  in  this 
serum.  But  when  introduced  beneath  the  skin  of  an  immune  rabbit, 
the  micrococcus  quickly  loses  its  virulence.  At  the  end  of  eighteen 
hours  it  has  completely  lost  its  pathogenic  power,  and  cultures  made 
in  bouillon  no  longer  have  any  injurious  effect  upon  rabbits.  This 
attenuating  effect  produced  in  the  body  of  an  immune  animal  is 
ascribed  by  Issaeff  to  the  action  of  phagocytes,  which  are  said  to  be 
very  numerous,  and  in  the  course  of  five  or  six  hours  to  pick  up  all  of 
the  cocci  in  the  vicinity  of  the  point  of  inoculation.  These  are  not, 
however,  immediately  destroyed  in  the  interior  of  the  phagocytes,  but 
preserve  their  vitality  for  nearly  forty -eight  hours,  and  when  intro- 
duced into  bouillon  give  a  culture  which  has  no  longer  any  patho- 
genic virulence. 

•  RINDERPEST. 

The  disease  of  cattle  known  in  Germany  as  rinderpest  is  due  to  a 
bacillus  closely  resembling  the  bacillus  of  fowl  cholera  and  of  swine 
plague  (BaciUus  septiccemice  hcemarrhagicce). 

Professor  Semmer,  of  St.  Petersburg,  has  reported  (1892)  his  suc- 
cess in  immunizing  cattle  against  this  disease.  The  virulence  of  cul- 
tures was  attenuated  by  passing  them  through  guinea-pigs,  or  by 
exposure  to  heat,  and  this  attenuated  virus  was  used  in  protective 
inoculations.  Semmer  says : 

"By  the  subcutaneous  injection  of  blood  serum  from  immune  animals 
their  susceptibility  to  rinderpest  was  diminished,  and  such  blood  serum  de- 
stroyed the  'rinderpest  contagium'  in  one  to  twenty-four  hours/' 

SWINE   PLAGUE. 

As  stated  in  the  chapter  on  cholera  in  fowls,  the  bacillus  of  swine 
plague  (ScJnveineseuche,  Loftier  and  Schiitz)  very  closely  resembles 
Pasteur's  microbe  of  fowl  cholera  and  Koch's  bacillus  of  rabbit  sep- 


344  PROTECTIVE   INOCULATIONS. 

ticsemia,  and  if  not  identical  with  these  at  least  varies  from  them  so 
slightly  in  its  morphological  and  biological  characters  that  recent 
authors  do  not  feel  justified  in  considering  it  a  distinct  species. 
Koch  first  obtained  his  bacillus  of  rabbit  septicaemia  by  inoculating 
rabbits  with  putrefying  flesh  infusion.  Gaffky  produced  the  same  in- 
fectious disease  in  rabbits  by  inoculating  them  with  impure  river  water. 
Davaine  had  previously  obtained  similar  results  by  inoculating  rabbits 
with  putrefying  blood.  The  writer  in  1887  produced  the  same  disease 
in  rabbits,  while  in  Cuba,  by  inoculating  them  with  putrefying  liver 
from  a  yellow-fever  cadaver.  A  similar,  and  possibly  identical,  ba- 
cillus has  been  found  in  the  blood  of  deer  (Hueppe),  of  cattle  (Kitt, 
and  of  buffalo  (Oreste-Armanni)  suffering  from  a  fatal  infectious  dis- 
ease. And  all  of  these  allied  species  or  varieties  are  included  by 
Hueppe  and  by  the  present  writer  under  the  single  specific  name 
Bacillus  septicwmice  hcemorrhagicw.  The  bacillus  of  the  disease  known 
in  this  country  as  swine  plague,  according  to  Smith,  agrees  in  all  par- 
ticulars with  that  of  the  German  swine  plague  (Sckweineseuche)  de- 
scribed by  Loftier  and  Schiitz,  except  that  the  latter  is  more  patho- 
genic for  swine  and  for  rabbits. 

In  a  publication  by  Smith  and  Moore  (United  States  Department 
of  Agriculture,  Bureau  of  Animal  Industry,  Bulletin  No.  6, 1894)  they 
have  given  an  account  of  their  experiments  relating  to  immunizing 
animals  against  the  pathogenic  action  of  this  bacillus.  The  bacilli 
used  in  these  experiments  were  sufficiently  virulent  to  kill  rabbits  in 
twenty  hours  when  injected  beneath  the  skin  of  these  animals  in  doses 
of  0.001  cubic  centimetre  of  a  fresh  bouillon  culture.  The  experiments 
were  made  upon  young  adult  rabbits  by  various  methods,  viz. :  with 
sterilized  bouillon  cultures ;  with  sterilized  suspensions  of  agar  cul- 
tures; with  the  filtrate  of  agar  suspensions;  with  defibrinated,  steril- 
ized blood  of  infected  rabbits ;  "with  blood  serum  from  immune  animals. 

"A  greater  or  less  degree  of  immunity  was  produced  in  rabbits  by  steril- 
ized bouillon  cultures,  sterilized  agar  suspensions,  sterilized  blood  from 
infected  rabbits,  and  blood  serum  from  immunized  rabbits.  The  sterilized 
blood  of  diseased  rabbits  was  capable  of  producing1  immunity,  while  the 
blood  serum  of  immune  rabbits  produced  rather  equivocal  results."1 

The  different  degrees  of  immunity  which  may  be  acquired  by 
rabbits,  as  shown  by  a  subsequent  inoculation  with  virulent  material, 
are  classified  by  Moore  as  follows : 

"1.  No  resistance — acute  septicaemia. 
"2.  Slight  resistance — peritonitis. 

"3.  Increased  resistance — pleuritis  and  pericarditis  with  or  without  sec- 
ondary pneumonia. 

"4.  Higher  degree  of  resistance — pleuritis  and  peritonitis. 


PROTECTIVE   INOCULATIONS.  345 

"5.  Still  greater  resistance — irregular  lesions  in  the  form  of  abscesses, 
subcutaneous  and  subperitoneal. 

4k6.  Nearly  complete  immunity — very  slight  reaction  at  the  point  of  in- 
oculation." 

Up  to  the  year  1894  the  bacteriological  experts  of  the  Depart- 
ment of  Agriculture  had  not  proposed  to  make  a  practical  application 
of  the  facts  developed  in  their  experimental  work  in  the  way  of  pro- 
tecting herds  of  swine  by  means  of  inoculations  with  an  attenuated 
virus,  or  with  sterilized  cultures.  In  the  report  on  swine  plague, 
made  by  the  Bureau  of  Animal  Industry  published  in  1891,  the  fol- 
lowing measures  for  arresting  an  epidemic  are  recommended : 

"When  the  disease  has  actually  appeared  in  a  herd  the  question  generally 
arises  whether  it  is  worth  while  to  make  any  attempt  to  save  a  portion  of  the 
herd  or  to  leave  them  to  their  fate.  As  a  rule  it  may  be  stated  that  it  is  best 
to  slaughter  both  healthy  and  diseased  at  once,  and  give  the  surroundings 
sufficient  time  to  rid  themselves  of  the  infection  before  fresh  animals  are 
brought  into  them.  If  this  be  not  desirable,  we  should  recommend  the  fol- 
lowing measures  to  be  vigorously  carried  out : 

"a.  Removal  of  still  healthy  animals  to  uninfected  grounds  or  pens  as 
soon  as  possible. 

"  b.  Destruction  of  all  diseased  animals. 

"  c.  Careful  burial  or  burning  of  carcasses. 

"d.  Repeated  thorough  disinfection  of  the  infected  premises. 

"e.  Great  cleanliness  both  as  to  surroundings  and  as  regards  food." 

In  the  same  report  (1891)  the  following  reference  is  made  to  pro- 
tective inoculations : 

"  As  regards  swine  plague  the  experiments  which  have  thus  far  been  car- 
ried out  indicate  that  this  disease  may  prove  amenable  to  preventive  inocula- 
tion. We  have  been  able,  by  the  injection  of  both  living  cultures  and  those 
sterilized  at  a  low  temperature  (58°  C.),  to  make  the  most  susceptible  animals 
— rabbits — insusceptible  to  the  most  virulent  swine  plague  bacteria.  By  two 
subcutaneous  injections  of  cultures  of  swine-plague  bacteria,  swine  have 
been  made  insusceptible  to  doses  injected  into  the  circulation  which  proved 
fatal  to  control  pigs  within  twenty-four  hours." 

According  to  Smith  the  experiments  of  Metchnikoff  (1892),  re- 
ported as  made  with  the  bacillus  of  hog  cholera,  were  in  fact  made 
with  the  bacillus  of  swine  plague ;  we  therefore  refer  to  them  here. 
These  experiments  showed  that  rabbits  could  be  easily  immunized 
against  the  pathogenic  action  of  virulent  cultures  by  means  of  blood, 
from  an  infected  animal,  sterilized  by  heat.  Doses  of  1.5  cubic  centi- 
metres, or  more,* were  fatal  to  rabbits;  but  smaller  doses,  repeated 
several  times,  given  either  subcutaneously  or  by  injection  into  the 
circulation,  caused  the  animal  to  become  immune. 

STREPTOCOCCUS   INFECTION. 

It  is  now  generally  recognized  by  pathologists  that  erysipelas, 
puerperal  fever,  certain  forms  of  diphtheritic  inflammation  of  the 


346  PROTECTIVE    INOCULATIONS. 

fauces,  and  certain  acute  abscesses  are  due  to  infection  by  a  strepto- 
coccus described  by  recent  authors  under  the  name  of  Streptococcus 
2)yogenes.  This  streptococcus,  like  other  pathogenic  microorganisms 
of  the  same  class,  varies  greatly  in  its  pathogenic  power  as  a  result  of 
conditions  relating  to  the  source  of  the  particular  variety  under  culti- 
vation. As  obtained  from  a  case  of  erysipelas  or  puerperal  fever  it  is 
extremely  virulent,  but  when  it  has  led  a  saprophytic  existence  for 
some  time,  or  has  been  cultivated  for  a  considerable  time  in  the 
usual  artificial  culture  media,  its  pathogenic  potency  is  greatly 
diminished. 

Mironoff  (1893)  has  made  a  series  of  experiments  with  a  view  to 
determining  whether  rabbits  can  be  immunized  against  the  pathogenic 
action  of  this  streptococcus,  and  has  obtained  successful  results  by  the 
following  method : 

Vigorous  rabbits,  weighing  two  kilogrammes,  were  inoculated 
subcutaneousl}'  with  from  three  to  six  cubic  centimetres  of  a  sterilized 
bouillon  culture  of  the  streptococcus.  Cultures  three  days  old  were 
employed,  and  these  were  sterilized  for  twenty  minutes  at  120°  C..— 
the  reason  for  using  so  high  a  temperature  is  not  apparent,  inasmuch 
as  this  streptococcus  is  destroyed  in  a  few  minutes  by  a  temperature 
of  60°  C.  At  the  end  of  ten  to  fifteen  days,  "  when  the  animal  has 
fully  recovered, "  a  second  dose  of  from  six  to  twelve  cubic  centimetres 
of  a  culture,  sterilized  in  the  same  way,  is  injected  beneath  the  skin. 
After  another  interval  of  ten  to  fifteen  days  two  cubic  centimetres  of  a 
virulent  non-sterilized  culture  are  injected  subcutaneously,  and  this  is 
repeated  with  gradually  increasing  doses  (one  to  two  cubic  centime- 
tres more)  at  intervals  of  the  same  period.  Finally  the  animals 
"  support  without  reaction  "  a  dose  five  times  as  great  as  would  be  re- 
quired to  kill  an  animal  of  the  same  weight  not  immunized.  But  the 
author  adds  that  more  than  half  the  animals  thus  treated  died  before 
the  completion  of  the  immunizing  process.  These  deaths  resulted 
from  local  infectious  processes,  such  as  peritonitis,  pericarditis,  men- 
ingitis, or  abscesses  formed  at  the  point  of  inoculation. 

Further  experiments  showed  that  the  blood  serum  of  animals  im- 
munized in  this  way  when  injected  into  susceptible  animals  (rabbits) 
in  the  dose  of  1.5  cubic  centimetres  per  kilogramme  of  body  weight 
conferred  upon  them  a  certain  degree  of  immunity  against  streptococ- 
cus infection,  and  with  twice  this  amount  (three  cubic  centimetres)  a 
very  decided  immunity  was  produced.  The  blood  serum  of  immune 
rabbits  in  doses  of  three  to  four  cubic  centimetres  per  kilogramme  of 
body  weight  was  found  to  exercise  a  curative  power,  and  completely  to 
arrest  the  acute  septicaemia  resulting  from  inoculations  with  a  virulent 


PROTECTIVE    INOCULATIONS.  347 

culture  of  this  streptococcus,  or  to  cause  the  disease  to  run  a  chronic 
course,  with  formation  of  abscesses  and  final  recovery. 

In  this  connection  we  may  call  attention  to  the  experiments  of 
Emmerich  (1886),  which  show  that  the  fatal  course  of  anthrax  infec- 
tion, in  rabbits,  may  be  arrested  by  the  subcutaneous  or  intravenous 
injection  of  this  streptococcus.  Subsequent  experiments  by  Emmerich 
and  de  Mattei  (1887)  showed  that  eleven  hours  after  such  an  injection 
the  anthrax  bacilli  were  all  dead  and  were  already  undergoing  degen- 
erative changes. 

Emmerich  and  his  associates  (1894)  have  reported  numerous  addi- 
tional experiments  which  show  that  the  blood  serum  of  a  rabbit  which 
is  suffering  from  streptococcus  septicaemia  (third  day),  when  filtered 
through  a  Pasteur-Chamberland  filter  to  remove  all  living  cocci,  may 
be  used  with  success  in  arresting  anthrax  infection  in  rabbits.  The 
filtered  serum  was  given  four  hours  after  the  anthrax  infection  in  the 
dose  of  twenty-five  cubic  centimetres  in  the  peritoneal  cavity  and  fif- 
teen cubic  centimetres  subcutaneously.  This  was  repeated  the  fol- 
lowing da3T  at  nine  o'clock  in  the  morning  and  five  o'clock  in  the 
evening,  and  again  on  the  third  day  in  the  morning.  Favorable  re- 
sults were  also  obtained  by  using  in  the  same  way  blood  serum  from 
a  sheep  infected  with  the  streptococcus. 

Cobbett  (1894)  reports  success  in  immunizing  rabbits  by  means  of 
attenuated  varieties  of  the  streptococcus  or  by  filtered  cultures.  Also 
that  cutaneous  erysipelas,  produced  by  inoculation,  after  recovery 
leaves  the  patient  immune  from  a  repetition  of  the  local  inflammatory 
process  as  a  result  of  a  subsequent  inoculation,  and  also  confers  a  gen- 
eral immunity  against  streptococcus  infection.  But  this  immunity  is 
of  short  duration,  not  lasting  longer  than  a  few  weeks.  Inoculation 
in  the  ear  of  a  rabbit,  protected  by  a  previous  inoculation  in  the  same 
locality,  is  followed  by  an  inflammatory  reaction ;  but  this  is  of  brief 
duration  and  has  disappeared  before  the  erysipelatous  inflammation 
produced  in  a  control  is  well  under  way. 

SYMPTOMATIC   ANTHRAX. 

This  disease  of  cattle  is  popularly  known  as  "  black  leg, "  or  "  quar- 
ter evil,"  and  is  described  by  German  authors  under  the  name  of 
Raitschbrand — French,  "  cltarboii  syiiiptomatiqm."  The  disease  pre- 
vails during  the  summer  months  in  various  parts  of  Europe,  and  to 
some  extent  in  the  United  States.  It  is  characterized  by  the  appear- 
ance of  irregular,  emphysematous  swellings  of  the  subcutaneous  tis- 
sues and  muscles,  especially  over  the  quarters.  The  muscles  in  the 


348  PROTECTIVE   INOCULATIONS. 

affected  areas  Lave  a  dark  color  and  contain  a  bloody  serum  in  which 
the  bacillus  is  found  to  which  the  disease  is  due.  This  is  an  an- 
aerobic bacillus  which  forms  large  oval  spores. 

The  etiology  of  the  disease  was  first  clearly  established  by  the  re- 
searches of  Arloing,  Cornevin,  and  Thomas  (1880  to  1883),  and  sub- 
sequent researches  have  shown  that  immunity  may  be  produced  in 
susceptible  animals  by  protective  inoculations. 

The  disease  causes  considerable  losses  among  cattle  in  certain  sec- 
tions. Horses  do  not  contract  it  spontaneously,  and  when  inoculated 
with  a  culture  of  the  bacillus  present  only  a  limited  local  reaction. 
Swine,  dogs,  rabbits,  fowls,  and  pigeons  have  but  slight  susceptibility. 
The  researches  of  the  authors  above  mentioned  have  shown  that  the 
virulence  of  a  culture  is  greatly  increased  by  adding  to  it  twenty  per 
cent  of  lactic  acid.  The  guinea-pig  is  the  most  susceptible  animal, 
and  succumbs  in  from  twenty-four  to  thirty -six  hours  when  inoculated 
subcutaneously  with  a  small  quantity  of  a  pure  culture.  According 
to  Kitasato  cultures  in  a  bouillon  made  from  the  flesh  of  the  guinea- 
pig  soon  lose  their  virulence,  while  cultures  in  solid  media  preserve 
their  virulence  for  a  long  time.  Cultures  are  readily  attenuated  by 
heat,  according  to  the  method  of  Toussaint  and  Chauveau — exposure 
to  a  temperature  of  42°  to  43°  C.  in  the  absence  of  spores.  The 
spores  are  attenuated  by  exposure  for  several  hours  to  a  temperature 
of  80°  to  100°  C.  Arloing,  Cornevin,  and  Thomas  recommend  for  the 
production  of  immunity  in  cattle  inoculation  with  a  dried  powder  of 
the  muscles  of  animals  recently  dead  from  the  disease.  This  is  at- 
tenuated by  heat.  According  to  Kitt  the  muscles  should  first  be 
dried  at  32°  to  35°  C.  and  then  powdered.  Two  "  vaccines  "  are  pre- 
pared from  this  powder — a  strong  vaccine  by  exposure  to  a  temper- 
ature of  85°  to  90°  C.  for  six  hours,  and  a  weaker  vaccine  by  exposure 
for  the  same  time  to  a  temperature  of  100°  to  104°  C.  (dry  heat).  An 
inoculation  is  first  made  with  the  weaker  vaccine  which  gives  rise  to 
a  local  reaction  of  moderate  intensity.  Later  a  second  inoculation  is 
made  with  the  stronger  vaccine,  after  which  the  animal  is  immune 
from  the  pathogenic  action  of  the  most  virulent  material.  Immunity 
may  also  be  secured  by  intravenous  injections ;  or,  in  guinea-pigs,  by 
inoculations  with  cultures  which  have  become  attenuated  by  being  kept 
a  few  days,  or  by  exposure  to  a  temperature  of  42°  to  43°  C. ;  or  by 
inoculation  with  a  very  small  quantity  of  a  pure  culture ;  or  by  inocu- 
lations with  filtered  cultures  (Eoux  and  Chamberland) ;  or  with  cultures 
sterilized  by  heat  (Kitasato).  A  non-fatal  and  protectivejocal  infec- 
tion may  also  be  produced  in  cattle  by  inoculations  with  virulent  ma- 
terial made  into  the  extremity  of  the  tail.  Eoux  has  claimed  that 


PROTECTIVE   INOCULATIONS.  349 

animals  which  have  an  acquired  immunity  against  symptomatic  an- 
thrax are  also  immune  against  the  pathogenic  action  of  the  bacillus  of 
malignant  oedema;  but  Kitasato  was  unable  to  confirm  this. 

Strebel,  in  1885,  published  the  results  of  protective  inoculations 
made  in  Switzerland  in  1884.  The  inoculations  were  made  in  the  end 
of  the  tail  with  two  "vaccines,"  with  an  interval  between  the  two  of 
from  nine  to  fourteen  days.  The  vaccines  were  prepared  by  exposure 
to  heat,  as  above  recommended  by  Arloing,  Cornevin,  and  Thomas. 
The  most  favorable  season  for  inoculations  was  found  to  be  the  spring, 
and  the  most  favorable  age  of  cattle  for  inoculation  from  five  months 
to  two  years. 

In  seven  Swiss  cantons  2,199  cattle  were  inoculated;  1,810  inocu- 
lations were  made  among  animals  which  were  exposed  in  dangerously 
infected  pastures.  Of  these  but  2  died,  one  two  months  and  the 
other  four  months  after  the  protective  inoculations.  Among  908  in- 
oculated cattle,  which  were  pastured  with  1,650  others  not  inoculated, 
the  mortality  was  0.22  per  cent,  while  the  loss  among  the  latter  was 
6.1  per  cent.  The  following  year  (1885),  according  to  Strebel,  the 
number  of  inoculations,  exclusive  of  those  made  in  the  canton  of  Bern, 
w;ts  35,000.  The  losses  among  inoculated  animals  are  reported  as  hav- 
ing been  about  five  times  less  than  among  those  not  protected  in  this 
way.  In  the  canton  of  Bern,  in  the  same  year,  according  to  Hess, 
15,137  cattle  were  inoculated  by  thirt}'-eight  veterinarians — 12,190  of 
these  were  pastured  in  dangerously  infected  pastures.  The  results 
are  said  to  have  been  favorable  to  the  method,  but  the  abstract  at 
hand  does  not  give  the  precise  figures. 

In  1887  Kitt  reported  the  results  of  his  investigations,  which  were 
confirmatory  of  those  previously  published  by  Arloing,  Cornevin, 
and  Thomas,  and  also  of  a  new  method  of  inoculation,  which  pre- 
sented the  advantage  that  a  single  inoculation  was  sufficient  to  confer 
immunity.  This  was  made  in  the  region  of  the  shoulder  with  a  vac- 
cine somewhat  stronger  than  that  employed  by  the  French  bacteriol- 
ogists, but  which  was  found  to  be  without  danger  for  cattle.  It 
produced  only  a  slight  local  effect.  His  vaccine  was  prepared  by 
heating  the  moistened  flesh  of  an  animal  just  dead  from  the  disease 
to  85°  to  90°  C.  for  six  hours.  This  did  not  kill  the  spores  present, 
but  caused  a  sufficient  attenuation  in  their  virulence. 

In  a  later  communication  (1888)  Kitt  recommends  that  the  flesh  of 
the  diseased  animal  be  first  dried  and  pulverized,  and  then  subjected 
to  a  temperature  of  100°  C.  in  streaming  steam  for  six  hours,  after 
which  it  is  to  be  again  dried  and  used  for  subcutaneous  inoculations. 
The  dose  is  from  five  to  fifteen  centigrammes. 


350  PROTECTIVE   INOCULATIONS. 

Roux  (1888)  lias  shown  by  experiment  that  sterilized  cultures  of 
the  bacillus,  which  have  been  exposed  to  a  temperature  of  115°  C., 
when  injected  in  doses  of  forty  cubic  centimetres,  three  times  repeated, 
into  the  cavity  of  the  abdomen  of  guinea-pigs,  cause  these  animals  to 
be  completely  immune  against  the  most  virulent  material.  Cultures 
from  which  the  bacilli  have  been  separated  by  filtration  are  still  more 
active.  And  immunity  could  easily  be  conferred  by  the  subcutaneous 
inoculatioD,  in  guinea-pigs,  of  one  cubic  centimetre  of  the.  filtrate  from 
the  serum  obtained  from  the  cedematous  tissues  of  a  diseased  animal. 

Schuhanka  (1888)  has  reported  the  results  of  inoculations  made 
in  the  dukedom  of  Salzburg  during  the  year  1887.  In  all  2,596  cattle 
were  inoculated  once,  and  2,472  twice,  with  an  attenuated  virus,  in 
forty -seven  different  parishes.  Most  of  these  were  from  six  months 
to  a  year  old.  No  losses  occurred  as  a  result  of  the  inoculations. 
During  the  summer  of  1887  the  2,472  cattle  which  had  been  twice  in- 
oculated were  associated  in  infected  pastures  with  3,561  unprotected 
cattle.  The  loss  among  the  former  was  8,  =  0.32  per  cent;  among  the 
latter  it  was  235,  =  6.31  per  cent. 

Strebel  reports  similar  results,  in  1887,  in  the  canton  Freiburg, 
where  1,725  cattle  which  had  been  inoculated  suffered  a  loss  of  0.23 
per  cent,  and  1,945  associated  cattle  a  loss  of  5.28  per  cent. 

Lydtin  (1892)  reports  the  results  of  inoculations  made  in  five  dis- 
tricts (Amtsbezirken)  in  Baden  during  the  years  1886-91:  2,797  cattle 
were  inoculated  with  a  loss  of  3  only  as  a  result  of  the  inoculation. 
None  of  the  inoculated  cattle  subsequently  contracted  the  disease. 

In  the  Bulletin  of  the  Central  Society  of  Veterinary  Medicine  of 
France  (1892),  Guillod  and  Simon  give  the  results  of  3,500  inocula- 
tions made  since  1884.  The  mortality  among  cattle  in  the  region 
where  these  inoculations  were  practised  had  been  from  10  to  20  per 
cent,  but  fell  to  0.5  per  cent  among  the  inoculated  animals. 

The  authors  last  named  prefer  inoculations  in  the  region  of  the 
shoulder  to  the  plan  first  practised  of  inoculating  in  the  end  of  the 
tail.  Strebel  also  (1892 )  advocates  this  method,  which  is  quickly  car- 
ried out  and  attended  with  but  little  loss.  According  to  Strebel  the 
loss  among  13,022  inoculated  in  this  way  only  amounted  to  5,  while 
the  loss  among  animals  inoculated  by  the  old  method  was  twice  as 
great. 

TETANUS. 

The  experiments  of  Kitasato  (1889)  show  that  pure  cultures  of  the 
tetanus  bacillus  injected  into  mice,  rabbits,  or  guinea-pigs  produce 
typical  tetanic  symptoms  and  death.  As  the  presence  of  this  bacillus 


PROTECTIVE    INOCULATIONS.  351 

at  the  seat  of  injury,  in  cases  of  tetanus  in  man,  lias  now  been 
demonstrated  by  numerous  observers,  there  is  no  longer  any  ques- 
tion that  tetanus  must  be  included  among  the  traumatic  infectious 
diseases,  and  that  the  bacillus  of  Nicolaier  and  of  Kitasato  is  the 
specific  infectious  agent.  Kitasato 's  experiments  (1890)  show  that 
cultures  of  the  tetanus  bacillus  which  have  been  sterilized  by  fil- 
tration through  porcelain  produce  the  same  symptoms,  and  death,  in 
the  animals  mentioned,  as  result  from  inoculation  with  cultures  con- 
taining the  bacillus.  It  is  evident,  therefore,  that  death  results  from 
the  action  of  a  toxic  substance  produced  by  the  bacillus.  This  is 
further  shown  by  the  fact  that  the  bacillus  itself  cannot  be  obtained  in 
cultures  from  the  blood  or  organs  of  an  animal  which  has  succumbed 
to  an  experimental  inoculation  with  an  unfiltered  culture ;  but  the 
blood  of  an  animal  killed  by  such  an  inoculation  contains  the  tetanus 
poison,  and  when  injected  into  a  mouse  causes  its  death  with  tetanic 
symptoms. 

When  a  platinum  needle  is  dipped  into  a  pure  culture  of  the  teta- 
nus bacillus,  and  a  mouse  is  inoculated  with  it  subcutaneously,  the 
animal  invariably  falls  sick  within  twenty-four  hours  and  dies  of 
typical  tetanus  in  two  or  three  days.  Rats,  guinea-pigs,  and  rabbits 
are  killed  in  the  same  way  by  somewhat  larger  quantities — 0.3  to  0.5 
cubic  centimetre  (Kitasato).  Pigeons  are  very  slightly  susceptible. 
The  tetanic  symptoms  are  first  developed  in  the  vicinity  of  the  point 
of  inoculation :  if  the  animal  is  inoculated  in  the  posterior  portion  of 
the  body,  the  hind  legs  first  show  tetanic  contraction;  if  in  the  fore- 
part of  the  body,  the  muscles  of  the  neck  are  first  affected.  At  the 
autopsy  there  is  a  certain  amount  of  hypersemia  at  the  point  of  inocu- 
lation, but  no  pus  is  formed;  in  inoculations  with  garden  earth,  or 
accidental  inoculations  in  man,  pus  is  commonly  found  in  the  vicinity 
of  the  inoculation  wound.  The  various  organs  are  normal  in  appear- 
ance. Kitasato  says  that  Jie  has  not  been  able  to  demonstrate  the 
presence  of  the  bacillus  or  of  spores  in  the  spinal  marrow,  .the  nerves, 
muscles,  spleen,  liver,  lungs,  kidneys,  or  blood  from  the  heart;  nor 
has  he  been  able  to  obtain  cultures  from  the  various  organs.  In  mice 
which  were  inoculated  at  the  root  of  the  tail  Kitasato  was  able  to  de- 
monstrate the  presence  of  the  bacilli  at  the  point  of  inoculation  by  the 
microscopical  examination  of  an  excised  piece  of  the  tissues  for  eight 
to  ten  hours  after  the  inoculation ;  later  than  this  they  were  not  found. 
In  pus  from  the  inoculation  wounds  of  men  and  animals  accidentally 
infected  the  bacilli  are  present,  but  the  formation  of  spores  does  not 
always  occur.  According  to  Kitasato,  the  sooner  death  has  occurred 
after  accidental  inoculation  the  less  likely  are  spores  to  be  found  in 


352  PROTECTIVE   INOCULATIONS. 

the  rods,  but  from  pus  in  which  no  spores  are  seen  cultures  of  the 
bacillus  may  be  obtained  in  which  spores  will  develop  in  the  usual 
manner. 

Guinea-pigs  are  even  more  susceptible  to  the  tetanus  poison  than 
mice,  and  rabbits  less  so.  The  amount  of  filtrate  from  a  slightly 
alkaline  bouillon  culture  required  to  kill  a  mouse  is  extremely  minute 
— 0.00001  cubic  centimetre  (Kitasato).  The  tetanic  symptoms  are 
developed  within  three  days;  if  the  animal  is  not  affected  within  four 
days  it  escapes  entirely.  The  tetanus  poison  is  destroyed  by  a  tem- 
perature of  65°  C.  maintained  for  five  minutes,  or  60°  for  twenty 
minutes,  or  55°  for  an  hour  and  a  half;  in  the  incubating  oven  at  37° 
C.  it  gradually  loses  its  toxic  potency ;  in  diffuse  daylight,  also,  its 
toxic  power  is  gradually  lost ;  in  a  cool,  dark  place  it  retains  its  origi- 
nal potency  indefinitely ;  in  direct  sunlight  it  is  completely  destroyed 
in  from  fifteen  to  eighteen  hours ;  it  is  not  injured  by  being  largely 
diluted  with  distilled  water;  it  is  destroyed  in  an  hour  by  hydro- 
chloric acid  in  the  proportion  of  0.55  per  cent;  terchloride  of  iodine 
destroys  it  in  the  proportion  of  0.5  per  cent;  cresol  in  one  per  cent — 
one  hour's  exposure.  In  general  it  is  destroyed  by  acids  and  by 
alkalies.  Blood  serum  from  cattle,  horses,  sheep,  rabbits,  rats,  or 
guinea-pigs  does  not  modify  its  toxic  properties. 

Brieger  (1886)  first  succeeded  in  obtaining  from  impure  cultures 
of  the  tetanus  bacillus  a  crystallizable  toxic  substance,  called  by  him 
tetanin,  which  was  found  to  kill  small  animals  in  very  minute  doses 
and  with  the  characteristic  symptoms  of  tetanus.  More  recently  Kit- 
asato and  Weyl  have  obtained  the  same  substance,  by  following 
Brieger's  method,  from  a  pure  culture  of  this  bacillus.  From  a  bouil- 
lon made  from  one  and  one-fourth  kilogrammes  of  lean  beef,  with  the 
addition  of  twenty -five  grammes  of  peptone,  they  obtained  1.7118 
grammes  of  hydrochlorate  of  tetauin.  This  proved  fatal  to  white  mice 
in  six  hours  in  the  dose  of  0.05  gramme,  and  a  dose  of  0.105  gramme 
caused  characteristic  tetanic  convulsions  and  death  within  an  hour. 
The  bacteriologists  last  named  also  obtained  from  their  cultures  the 
tetanotoxin  of  Brieger.  Two  mice  were  inoculated  subcutaneous!}' 
with  0.003  gramme  of  this  substance;  one  died  at  the  end  of  five 
hours  without  the  development  of  tetanic  symptoms ;  the  other  sur- 
vived. In  addition  to  these  substances,  indol,  phenol,  and  buty- 
ric acid  were  demonstrated  to  be  present  in  cultures  of  the  tetanus 
bacillus. 

The  more  recent  researches  of  Brieger  and  Frankel,  and  of  Kita- 
sato, show  that  the  toxic  ptomain  discovered  by  Brieger  in  1886  is 
not  the  substance  to  which  cultures  of  the  tetanus  bacillus  owe  their 


PROTECTIVE   INOCULATIONS.  353 

great  and  peculiar  pathogenic  power.  The  distinguished  German 
chemist  and  his  associate  have  succeeded  in  isolating  from  tetanus 
cultures  a  toxalbumin  which  is  far  more  deadly  than  tetanin. 

Brieger  and  Cohn  in  more  recent  investigations  (1893)  relating  to 
the  toxic  products  of  the  tetanus  bacillus  have  arrived  at  the  following 
results :  The  cultures  were  made  in  veal  bouillon  containing  one  per 
cent  of  peptone  and  one-fifth  per  cent  of  chloride  of  sodium.  Large 
quantities  of  the  cultures  in  this  medium  were  filtered  through  porce- 
lain filters.  The  active  substance  was  precipitated  from  the  filtrate 
by  means  of  a  saturated  solution  of  ammonium  sulphate.  By  adding 
this  salt  in  excess  the  precipitate  is  made  to  rise  to  the  surface  and  is 
skimmed  off  with  a  platinum  spatula.  The  liquid  is  removed  by  plac- 
ing this  upon  porous  porcelain  plates  and  the  crude  toxin  is  dried  in 
a  vacuum.  It  still  contains  6.5  per  cent  of  ammonium  sulphate.  The 
tetanus  bouillon  after  filtration  is  said  to  be  fatal  to  mice  in  the  dose 
of  0.00005  cubic  centimetre.  A  litre  of  this  bouillon  gave  about  one 
gramme  of  the  dried  precipitate,  which  produced  characteristic  te- 
tanic symptoms  and  death  when  injected  into  mice  in  the  dose  of 
0.0000001  gramme.  Kitasato  in  his  experiments  had  previously  ob- 
tained a  tetanus  bouillon  which  was  five  times  as  toxic  as  that  used  by 
Brieger  and  Cohn  in  their  experiments,  and  which  killed  mice  in  the 
dose  of  0.00001  cubic  centimetre.  The  dried  precipitate  obtained  by 
Brieger  and  Cohn  contained  various  impurities,  including  a  certain 
amount  of  ammonium  sulphate,  but  was  found  to  kill  susceptible  ani- 
mals in  the  proportion  of  0.0000066  gramme  per  kilogramme  of  body 
weight. 

It  was  purified  without  loss  of  toxic  power  by  placing  it  in  a  dialyzer 
in  running  water  for  from  twenty -four  to  f  orb* -eight  hours,  after  which 
it  was  dried  in  vacua  at  20°  to  22°  C.  The  purified  toxin  thus  ob- 
tained had  a  slightly  yellowish  color,  and  was  in  the  form  of  trans- 
parent scales,  which  were  odorless,  tasted  like  gum  acacia,  and  were 
easily  soluble  in  water.  The  chemical  reactions  of  this  purified  toxin, 
according  to  Brieger  and  Cohn,  show  that  it  is  not  a  true  albuminous 
body.  When  injected  beneath  the  skin  of  a  mouse  weighing  fifteen 
grammes,  in  the  dose  of  0.00000005  gramme,  it  causes  its  death,  and 
one-fifth  of  this  amount  gave  rise  to  tetanic  symptoms  from  which  the 
animal  recovered  after  a  time.  The  lethal  dose  for  a  man  weighing 
seventy  kilogrammes  is  estimated  by  the  authors  named  to  be  0.00023 
gramme  (0.23  milligramme).  Comparing  this  with  the  most  deadly 
vegetable  alkaloids  known  it  is  nearly  six  hundred  times  as  potent  as 
atropine  and  one  hundred  and  fifty  times  as  potent  as  strychnine. 

Fermi  and  Pernossi  (1894),  as  a  result  of  an  elaborate  research, 
23 


354  PROTECTIVE   INOCULATIONS. 

have  determined  many  of  the  chemical  characters  of  the  tetanus  toxin. 
When  in  solution  it  is  destroyed  by  a  comparatively  low  temperature 
(55°  C.  for  one  hour)  and  by  exposure  to  direct  sunlight,  but  the  dry 
powder  resists  a  temperature  of  120°  C.  It  has  not  the  properties  of 
an  alkaloid,  as  it  is  not  dissolved  by  any  of  the  usual  solvents  of  these 
bodies — the  only  solvent  thus  far  discovered  is  said  to  be  water.  It 
resembles  the  albumins  and  peptones  in  its  failure  to  pass  through 
a  dialyzing  membrane.  The  authors  last  referred  to  conclude  their 
summary  of  results  as  follows : 

"The  appended  table  shows  that  the  tetanus  poison,  like  that  of  diph- 
theria, in  its  behavior  as  regards  the  action  of  light,  heat,  chemical  agents, 
and  dialysis,  as  also  its  solvents,  the  agents  which  precipitate  it,  and  its  action 
upon  living  animals,  closely  resembles  the  poisons  of  serpents  (Naja  tripu- 
dians,  Crotalus,  etc.).  As  to  the  chemical  nature  of  this  group  of  substances, 
we  can  at  present  only  say  that  they  rather  have  the  characters  of  collodial 
substances  than  otherwise,  and  more  nearly  resemble  the  albuminoid  bodies 
ihan  the  bases.  We  do  not,  however,  reject  the  very  probable  hypothesis 
that  these  toxins  are  acids  or  bases,  or  other  very  unstable,  peculiar  substances, 
which  are  closely  united  with  colloidal  substances,  as  is  the  case,  for  example, 
with  the  alkali  and  acid  albumins  and  so  many  other  albuminous  bodies." 

While  the  exact  nature  of  the  toxic  substance  contained  in  tetanus 
cultures  has  not  been  determined,  we  probably  cannot,  at  present,  do 
better  than  to  continue  to  speak  of  it  as  a  "toxalbumin." 

Kitasato  (1891)  was  not  able  to  produce  immunity  in  mice  by  in- 
oculations with  minute  doses  of  the  poison,  or  with  a  filtrate  which 
had  been  exposed  to  various  degrees  of  temperature  by  which  its 
activity  was  diminished  or  destroyed.  But  immunity  lasting  for 
about  two  months  was  produced  in  rabbits  by  inoculating  them  with 
the  filtrate  from  a  culture  of  the  tetanus  bacillus,  and  subsequently, 
in  the  same  locality,  with  three  cubic  centimetres  of  a  one-per-cent 
solution  of  terchloride  of  iodine;  this  last  solution  was  injected  sub- 
cutaneously  in  the  same  dose  at  intervals  of  twenty -four  hours  for  five 
days.  Of  fifteen  rabbits  treated  in  this  way  six  proved  to  be  immune 
against  large  doses  of  a  virulent  culture  of  the  tetanus  bacillus.  The 
same  treatment  was  not  successful  in  producing  immunity  in  mice  or 
guinea-pigs,  but  the  important  discovery  was  made  that  a  small  quan- 
tity of  blood  (0.2  cubic  centimetre)  from  an  immune  rabbit,  when  in- 
jected into  the  abdominal  cavity  of  a  mouse,  gave  it  immunity  from 
the  effects  of  inoculations  with  the  tetanus  bacillus.  Moreover,  mice 
which  were  first  inoculated  with  a  virulent  culture  of  the  bacillus,  and, 
after  tetanic  symptoms  had  appeared,  received  in  the  cavity  of  the 
abdomen  an  injection  of  blood  serum  from  an  immune  mouse,  were 
preserved  from  death.  The  power  of  the  blood  of  an  immune  animal 
to  neutralize  the  tetanus  poison  was  further  shown  by  mixing  the  fil- 


PROTECTIVE   INOCULATIONS.  355 

trate  from  a  virulent  culture  with  blood  serum  from  an  immune  animal 
and  allowing  it  to  stand  for  twenty -four  hours;  a  dose  three  hundred 
times  greater  than  would  have  sufficed  to  kill  a  mouse  proved  to  be 
without  effect  after  such  admixtures  with  blood  serum;  as  before 
stated,  the  blood  serum  of  animals  which  are  not  immune  has  no 
effect  upon  the  poison.  The  duration  of  immunity  induced  in  this 
way  was  from  forty  to  fifty  days.  Blood  serum  from  an  immune  rab- 
bit, preserved  in  a  cool,  dark  room,  retains  its  power  of  neutralizing 
the  tetanus  poison  for  about  a  week,  after  which  time  it  gradually 
loses  it.  Having  found  that  chickens  have  a  natural  immunity  against 
tetanus,  Kitasato  made  experiments  to  ascertain  whether  their  blood 
serum  would  also  neutralize  the  tetanus  poison ;  the  result  was  nega- 
tive. 

That  the  tetanus  poison  is  present  in  the  blood  of  individuals  who 
die  from  tetanus  has  been  proved  by  Kitasato  by  injecting  a  small 
quantity  (0.2  to  0.3  cubic  centimetre)  of  blood  from  the  heart  of  a 
fresh  cadaver  into  mice;  the  animals  develop  typical  tetanic  symp- 
toms and  die  in  from  twenty  hours  to  three  days. 

Tizzoni  and  Cattani  have  (1891)  reported  results  similar  to  those 
obtain  by  Kitasato.  By  repeated  inoculations  with  gradually  in- 
creasing doses  of  the  tetanus  poison  they  succeeded  in  making  a  dog 
and  two  pigeons  immune,  and  found  that  blood  serum  from  this  im- 
mune dog,  in  very  small  amount,  completely  destroyed  the  toxic 
power  of  a  filtrate  from  cultures  of  the  tetanus  bacillus — one  to  two 
drops  of  serum  neutralized  0.5  cubic  centimetre  of  filtrate  after  fifteen 
to  twenty  minutes'  contact.  They  also  ascertained  that  small  amounts 
of  blood  serum  from  this  immune  dog  injected  into  other  dogs  or 
white  mice  produced  immunity  in  these  animals;  but  they  were  not 
able  to  produce  immunity  in  guinea-pigs  or  rabbits  by  the  same 
method. 

In  a  later  communication  (May,  1891)  Tizzoni  and  Cattani  give 
an  account  of  their  experiments  made  with  a  view  to  determining 
the  nature  of  the  substance  in  the  blood  serum  of  an  immune  animal 
which  has  the  power  of  destroying  the  toxalbumin  of  tetanus — "  tet- 
anus antitoxin."  They  found,  in  the  first  place,  that  this  antitoxin 
in  blood  serum  is  destroyed  in  half  an  hour  by  a  temperature  of  68° 
C. ;  further,  that  it  does  not  pass  through  a  dialyzing  membrane;  that 
it  is  destroyed  by  acids  and  alkalies.  As  a  result  of  their  researches 
they  conclude  that  it  is  an  albuminous  substance  having  the  nature  of 
an  enzyme. 

Yaillard  has  succeeded  in  producing  immunity  in  rabbits  by  re- 
peated injections  into  the  circulation  of  filtered  cultures — in  all  twenty 


350  PROTECTIVE   INOCULATIONS. 

cubic  centimetres — which  had  been  exposed  for  one  hour  to  a  temper- 
ature of  60°  C.  At  a  temperature  of  65°  C.  both  the  toxic  and  the 
immunizing  action  is  destroyed. 

Behring  (1892)  gives  the  following  account  of  a  method  which  he 
has  successfully  employed  for  producing  immunity  in  large  animals — 
especially  in  horses:  A  culture  of  the  tetanus  bacillus  is  made,  in 
bouillon,  of  such  toxic  potency  that  0.75  cubic  centimetre  will  kill  a 
rabbit  in  three  or  four  days.  To  two  hundred  cubic  centimetres  of 
this  culture  he  adds  carbolic  acid  in  the  proportion  of  0.5  per  cent  for 
the  purpose  of  preserving  it.  The  horse  first  receives  a  subcutaneous 
injection  of  ten  cubic  centimetres  of  this  culture  fluid  to  which  ter- 
chloride  of  iodine  (IC13)  has  been  added  in  the  proportion  of  0.25 
per  cent;  at  the  end  of  eight  days  twenty  cubic  centimetres  of  the 
same  mixture  are  given;  again  in  eight  days  the  dose  is  repeated; 
then,  after  an  interval  of  three  days,  thirty  cubic  centimetres  of  the 
same  mixture.  Following  this,  at  an  interval  of  eight  days,  he  gives 
two  injections  of  thirty  cubic  centimetres  each  of  a  mixture  containing 
one-half  the  quantity  of  IC13  (0.175  per  cent).  The  proportion  of 
the  iodine  terchloride  is  then  reduced  to  0.125  per  cent,  and  two 
doses  of  twenty  cubic  centimetres  each  are  given.  Finally  the  culture 
fluid  is  administered  in  the  dose  of  0.5  cubic  centimetre,  and  this  dose 
is  doubled  every  five  days.  Before  giving  the  first  dose  of  culture 
fluid  without  the  addition  of  IC13,  the  immunizing  value  of  the  blood 
serum  of  the  horse  is  tested  on  mice,  and  if  it  falls  below  1 : 100  a 
dose  of  0.25  cubic  centimetre  is  given  instead  of  the  larger  dose  (0.5 
cubic  centimetre)  above  mentioned. 

Schutz  (1892)  has  applied  Behring 's  method  to  a  considerable 
number  of  horses  and  sheep,  and  arrives  at  the  conclusion  that  it  is  a 
reliable  method  of  protecting  these  animals  against  infection  with  liv- 
ing tetanus  bacilli  and  against  the  toxic  action  of  filtered  cultures ; 
that  the  degree  of  immunity"  and  the  antitoxic  power  of  the  blood 
serum  increase  as  larger  doses  are  gradually  given.  According  to 
Behring  the  immunizing  value  of  blood  serum  from  a  horse  treated  in 
this  way  is  very  high.  As  tested  on  mice  it  may  be  1 : 200,000,  or  even 
more.  According  to  his  calculations  a  serum  having  a  value  of  1 : 100,- 
000,  as  tested  on  mice,  should  be  given  to  a  man  weighing  fifty  kilo- 
grammes in  the  quantity  of  fifty  cubic  centimetres,  given  in  the  course 
of  two  days,  in  order  to  insure  immunity. 

The  same  author  in  a  subsequent  paper  (1892)  gives  details  as  to 
the  method  of  estimating  the  therapeutic  value  of  serum  from  an  im- 
mune animal.  He  first  calls  attention  to  the  fact  that  the  only  re- 
agent by  which  the  antitoxic  potency  of  this  serum  can  be  tested  is 


PROTECTIVE   INOCULATIONS.  357 

the  body  of  a  living  animal.  The  test  animal  selected  is  the  white 
mouse.  When  the  statement  is  made  that  a  serum  has  the  value  of 
1 : 1,000,000,  he  means  that  by  an  experimental  test,  made  upon  white 
mice,  it  has  been  ascertained  that  these  animals  are  protected  from 
fatal  infection  with  the  minimal  lethal  dose  of  a  tetanus  culture  by  the 
use  of  0.00002  gramme  of  the  serum  for  a  mouse  weighing  twenty 
grammes.  For  the  cure  of  tetanus  in  the  mouse,  after  the  first  symp- 
toms of  the  disease  have  appeared,  a  dose  at  least  one  thousand  times 
as  great  as  the  immunizing  dose  is  required,  and  the  more  advanced 
the  progress  of  the  case  the  greater  the  dose  must  be.  A  serum  of 
the  strength  above  indicated,  if  used  for  the  treatment  of  a  case  of 
tetanus  in  man,  should,  according  to  Behring,  be  employed  in  doses 
amounting  altogether  to  at  least  one  hundred  cubic  centimetres — given 
inside  of  twenty -four  hours  in  doses  of  twenty  cubic  centimetres  each. 
For  persons  sixteen  years  old  he  would  give  doses  of  ten  cubic  centi- 
metres, and  for  children  under  six,  five  cubic  centimetres  at  a  dose. 
The  serum  of  this  strength  which  he  had  prepared  for  testing  its 
curative  value  on  man  was  preserved  by  the  addition  of  0.5  per  cent 
of  carbolic  acid. 

Rotter  (1892)  reports  a  case  successfully  treated  by  Behring 's 
serum.  In  all  two  hundred  and  fifty  cubic  centimetres  was  adminis- 
tered subcutaneously.  The  case  was  not,  however,  one  of  the  most 
severe  forms  of  the  disease. 

Brieger  and  Ehrlich  (1892)  have  succeeded  in  immunizing  goats 
by  means  of  gradually  increasing  doses  of  a  culture  of  the  tetanus 
bacillus  in  thymus  bouillon.  The  amount  given  at  first  was  0.2  cubic 
centimetre,  and  this  was  gradually  increased  to  ten  cubic  centimetres. 
At  the  end  of  thirty -seven  days  the  animal  was  found  to  be  immune 
against  virulent  cultures,  and  the  important  fact  was  demonstrated 
that  the  immunizing  substance  (antitoxin)  was  present  in  its  milk. 
A  mouse  which  received  0.1  cubic  centimetre  of  the  milk  of  this  goat 
in  the  peritoneal  cavity  proved  to  be  immune  against  infection  as  a 
result  of  inoculation  with  a  tetanus  culture.  The  immunizing  value 
of  the  milk  from  this  goat  was  found  to  be  1,600.  That  is,  a  dose  of 
0.2  cubic  centimetre,  which  was  equal  to  1 : 100  of  the  body  weight  of 
the  animal,  protected  a  mouse  from  sixteen  times  the  fatal  dose  of  a 
tetanus  culture.  After  precipitation  of  the  casein  the  milk  still  pre- 
served its  antitoxic  power  unimpaired,  and  by  concentrating  it  in  vacao 
a  fluid  was  obtained  which  proved  to  have  an  immunizing  value  of 
5,000. 

In  a  later  communication  (1893)  Brieger  and  Colin  give  the  results 
of  additional  experiments  with  the  milk  of  immunized  goats.  Ani- 


358  PROTECTIVE   INOCULATIONS. 

mals  were  chosen  which  were  two  or  three  years  old  and  had  given 
birth  to  young  a  few  weeks  before  the  inoculations  were  commenced. 
It  having  been  previously  shown  by  Ehrlich  that  the  precipitated 
tetanus  toxin  from  cultures  could  be  successfully  used  to  immunize 
guinea-pigs,  the  same  substance  was  employed  in  these  experiments. 
The  treatment  was  commenced  with  a  dose  of  0.00001  gramme,  which 
was  carefully  increased  to  0.00007  gramme,  the  injections  being  made 
at  intervals  of  four  days.  But  this  proved  to  be  too  much,  and  the 
animal  died  of  typical  tetanus  after  the  last  dose.  In  a  subsequent 
experiment  Brieger  and  Colin  succeeded  in  immunizing  a  goat  in  a 
month  and  a  half  so  that  the  animal  finally  withstood  a  dose  of  0.06 
gramme,  but  this  animal  ceased  to  give  milk,  became  anaemic,  and 
finally  died. 

The  authors  therefore  resorted  to  a  different  method  which  had 
previously  been  successfully  employed  by  Ehrlich,  Behring,  and 
others.  Cultures  of  the  tetanus  bacillus  in  bouillon  were  heated  to 
65°  C.  for  half  an  hour,  and  then  used  for  immunizing  two  goats. 
After  five  weeks'  treatment  the  animals  resisted  doses  of  the  precipi- 
tated toxin,  which  were  gradually  increased  to  ten  grammes,  at  which 
time  the  treatment  had  been  carried  on  for  nearly  six  months  and  the 
antitoxic  value  of  the  milk  was  found  to  be  90,000  immunization  units. 

The  method  of  determining  antitoxic  values  adopted  by  Brieger 
and  Cohn  is  the  following :  They  had  found  by  carefully  conducted 
experiments  that  their  precipitated  toxin  (Eohgifte)  killed  a  mouse 
weighing  twenty  grammes  in  the  dose  of  0.0000003  gramme,  but 
failed  to  kill  when  injected  in  the  dose  of  0.0000002  gramme.  The 
first-mentioned  dose  was  therefore  accepted  as  the  minimum  fatal  dose 
for  an  animal  weighing  eighteen  to  twenty  grammes,  and  the  object 
in  view  was  to  find  the  minimum  amount  of  milk  required  to  prevent 
the  toxic  action  of  such  a  dose. 

The  antitoxin  was  obtained  from  the  goat's  milk  by  precipitation 
with  ammonium  sulphate,  thirty -two  per  cent;  the  precipitate  was 
again  dissolved  and  treated  with  a  solution  of  basic  acetate  of  lead; 
this  salt  does  not  precipitate  the  antitoxin  when  the  solution  is  slightly 
alkaline;  the  voluminous  precipitate  produced  by  the  lead  acetate  is 
filtered  out  and  repeatedly  washed  with  water;  the  filtered  fluid  and 
wash  water  are  again  treated  with  ammonium  sulphate,  added  to 
saturation,  and  the  resulting  precipitate  is  dissolved  in  a  small  quan- 
tity of  water;  a  precipitate  is  again  obtained  by  saturation  with  am- 
monium sulphate,  and  this  is  dried  upon  porcelain  plates  in  a  vac- 
uum. The  ammonium  sulphate  remaining  could  not  be  removed  by 
dialysis,  as  experiment  showed  that  a  considerable  loss  of  the  antitoxin 


PROTECTIVE    INOCULATIONS.  359 

occurred  in  a  dialyzer  placed  in  running  water.  But  by  shaking  up 
the  dry  powder  in  chloroform  the  heavy  salt  sank  to  the  bottom  and 
the  purified  antitoxin  floated  on  the  surface  and  could  be  recovered  by 
skimming  it  off.  The  powder  thus  obtained  consisted  of  a  mixture  of 
various  substances,  including  the  antitoxin,  and  when  obtained  from 
milk  having  an  antitoxic  value  of  90,000  it  was  found  to  have  a  value 
of  25,000,000  immunization  units.  By  further  purification  a  still 
higher  value  was  obtained  (55,000,000).  In  experiments  on  mice  a 
dose  ten  thousand  times  as  great  as  was  necessary  to  produce  immu- 
nity proved  to  exercise  a  curative  power — i.e.,  a  dose  of  0.02  gramme 
for  a  mouse  weighing  twenty  grammes  saved  it  from  being  killed  by 
double  the  minimum  fatal  dose  of  the  tetanus  toxin,  after  tetanic 
symptoms  had  been  developed. 

Reference  has  been  made  to  the  production  of  immunity  by  the 
use  of  cultures  made  in  thymus  bouillon.  This  was  made  known 
through  the  experiments  of  Brieger,  Kitasato,  and  Wassermann 
(1892).  The  thymus  bouillon  is  made  from  the  thymus  glands  of 
calves,  which  are  chopped  fine  in  a  hash  machine  and  covered  with 
an  equal  volume  of  distilled  water.  The  mixture  is  stirred  for  some 
time  and  then  placed  in  an  ice  chest  for  twelve  hours;  the  liquid  is 
then  obtained  by  filtration  through  gauze  with  pressure — by  means  of 
a  flesh-press  machine.  A  turbid,  slimy  fluid  is  thus  obtained,  which 
is  diluted  with  an  equal  volume  of  water  and  made  slightly  alkaline 
by  the  addition  of  soda  solution.  It  is  then  sterilized  at  100°  C.  for 
fifteen  minutes.  As  a  result  of  this  the  liquid  has  a  grayish-brown 
color,  and  some  large  flocculi  in  suspension,  which  are  removed  by 
passing  it  through  fine  linen.  The  fluid  is  then  of  a  milky  opal- 
escence.  It  is  next  placed  in  test  tubes  and  again  sterilized.  The 
tetanus  bacillus  when  cultivated  in  this  medium  does  not  form  spores, 
and  the  toxic  potency  of  the  culture  is  very  much  reduced — 1 : 5,000  to 
1 : 3,000  of  the  toxic  potency  manifested  by  cultures  of  the  same  bacil- 
lus in  ordinary  media.  Inoculations  with  cultures  in  thymus  bouillon 
were  found  to  kill  mice  in  the  dose  of  0.5  cubic  centimetre,  while 
smaller  amounts  failed  to  kill  and  caused  the  animals  to  be  immune. 
A  culture  in  ordinary  bouillon  was  fatal  to  mice  in  the  dose  of  0.001 
cubic  centimetre. 

Experiments  on  rabbits  (thirty -five)  gave  a  uniformly  successful 
result  in  immunizing  these  animals.  Immunity  was  established  in 
the  course  of  two  weeks,  and  the  blood  serum  of  these  animals  tested 
on  mice  showed  an  antitoxic  value  of  1,000. 

Reference  has  already  been  made  to  the  earlier  researches  of  the 
Italian  investigators,  Tizzoni  and  Cattani.  These  have  been  followed 


360  PROTECTIVE   INOCULATIONS. 

by  additional  investigations,  the  results  of  which  have  been  reported 
in  numerous  published  papers.  The  authors  named  have  ascertained 
that  when  kept  in  a  cool  place  (15°  to  25°  C.)  the  blood  serum  of 
immune  rabbits  retains  its  antitoxic  power  for  several  months,  and 
the  antitoxin,  obtained  by  precipitation  with  alcohol,  kept  in  a  dry 
condition  for  more  than  ten  months,  was  found  to  preserve  its  original 
activity. 

Having  succeeded  in  their  earlier  experiments  in  immunizing  rab- 
bits and  dogs,  Tizzoni  and  Cattani  (in  1893)  proceeded  to  experiment 
upon  horses,  and  were  equally  successful  with  these  animals.  As  a 
result  of  numerous  injections  with  an  attenuated  virus,  continued  for 
a  period  of  ninety-seven  days,  they  established  an  immunity  which 
was  tested  by  inoculating  the  animal  with  ten  cubic  centimetres  of  a 
gelatin  culture,  of  which  one  two-hundredth  part  of  a  drop  killed  a 
white  mouse.  The  antitoxic  value  of  the  blood  serum  of  this  horse 
was  1 : 5, 000, 000 — i.e.,  one  gramme  of  this  serum  would  immunize  five 
million  grammes  of  mice,  or  two  hundred  and  fifty  thousand  mice 
weighing  twenty  grammes  each.  In  a  later  communication  (1894)  the 
authors  named  report  that  after  freely  bleeding  immunized  horses,  and 
allowing  them  to  rest  for  one  or  two  months,  and  then  again  treating 
them  with  small  doses  of  tetanus  cultures,  the  blood  serum  soon  be- 
comes as  active  as  before  the  bleeding.  The  greatest  antitoxic  power 
was  manifested  from  twenty  to  twenty-three  days  after  the  completion 
of  the  protective  inoculations,  and  a  serum  was  obtained  possessing  a 
value  of  1 : 10,000,000.  According  to  the  authors  named  the  precipi- 
tated (by  alcohol)  and  purified  antitoxin  from  such  a  serum,  judging 
from  their  experiments  on  lower  animals,  should  cure  a  case  of  teta- 
nus in  man  in  the  dose  of  from  forty  to  fifty  centigrammes. 

The  authors  last  mentioned  have  reported  (1892)  that  the  young  of 
immune  parents  have  a  certain  degree  of  inherited  immunity.  And 
the  more  recent  experiments  of  Ehrlich  and  Hubener  have  confirmed 
this  so  far  as  the  inheritance  of  immunity  from  the  mother  (in  mice) 
is  concerned;  but  their  results  did  not  show  any  immunity  in  the 
young  when  only  the  father  had  been  rendered  immune ;  and  the  im- 
munity inherited  from  the  mother  only  lasted  for  two  or  three  months 
after  birth. 

TUBERCULOSIS. 

Metchnikoff  states  that  when  kept  at  a  temperature  of  42°  C.  for 
some  time  the  tubercle  bacillus  undergoes  a  notable  diminution  in  its 
pathogenic  power,  and  that  when  kept  at  a  temperature  of  43°  to  44° 
C.  it  after  a  time  only  induces  a  local  abscess  when  injected  subcu- 


PROTECTIVE    INOCULATIONS.  361 

taneously  into  guinea-pigs.  The  experiments  of  Lote  also  indicate 
that  an  "attenuation  of  virulence"  has  occurred  in  the  cultures  pre- 
served in  Koch's  laboratory,  originating  in  1882  from  the  lungs  of  a 
tuberculous  ape.  The  author  named  made  experiments  with  cultures 
from  this  source  (ninetieth  to  ninety -fifth  successive  cultures),  and  at 
the  same  time  with  a  culture  obtained  from  Roux,  of  Pasteur's  labor- 
atory. Rabbits  inoculated  with  cultures  from  the  last-mentioned 
source  developed  a  hectic  fever  at  the  end  of  two  weeks,  and  died  tu- 
berculous at  the  end  of  twenty -one  to  thirty-nine  days.  Twelve  rab- 
bits were  inoculated  with  the  cultures  from  Koch's  laboratory ;  the 
injections  were  made  either  subcutaneously,  or  into  a  vein,  or  into  the 
pleural  cavity,  or  into  the  cavity  of  the  abdomen.  No  elevation  of 
temperature  occurred  in  any  of  the  animals,  and  they  were  found  at 
the  end  of  a  month  to  have  increased  in  weight.  At  the  end  of  six 
weeks  one  of  them  was  killed  and  tubercular  nodules  were  found  in 
various  organs.  The  remaining  animals  were  killed  at  the  end  of 
one  hundred  and  forty -four  to.  one  hundred  and  forty -eight  days. 
The  two  inoculated  subcutaneously  presented  no  sign  of  general  tu- 
berculosis, but  a  small  yellow  nodule  containing  bacilli  was  found  at 
the  point  of  inoculation.  Those  inoculated  by  injection  into  a  vein 
showed  one  or  two  nodules  in  the  lungs  containing  a  few  bacilli.  In 
Koch's  original  experiments  rabbits  were  killed  by  intravenous  inocu- 
lation of  his  cultures  in  from  thirteen  to  thirty-one  days.  That  this 
attenuation  of  virulence  depends  upon  a  diminished  production  of 
toxic  product  to  which  the  bacillus  owes  its  pathogenic  power  appears 
to  be  very  certain,  in  view  of  the  fact  that  the  late  cultures  in  a  series 
have  a  more  vigorous  and  abundant  development  than  the  more  patho- 
genic cultures  obtained  directly  from  the  animal  body. 

The  discovery  by  Koch  of  a  toxin  in  cultures  of  this  bacillus, 
which  is  soluble  in  glycerin,  and  which  in  very  minute  doses  pro- 
duces febrile  reaction  and  other  decided  symptoms  when  injected 
subcutaneously  into  tuberculous  animals,  must  rank  as  one  of  the  first 
importance  in  scientific  medicine,  whatever  the  final  verdict  may  be 
as  to  its  therapeutic  value  in  tuberculous  diseases  in  man. 

The  toxic  substance  contained  in  Koch's  glycerin  extract  from  cul- 
tures of  the  tubercle  bacillus,  now  generally  known  under  the  name  of 
tuberculin,  is  soluble  in  water,  insoluble  in  alcohol,  and  passes  readily 
through  dialyzing  membranes.  It  is  not  destroyed  by  the  boiling 
temperature.  According  to  the  chemical  examination  of  Jolles,  the 
"  lymph  "  contains  fifty  per  cent  of  water  and  does  not  contain  alka- 
loids or  cyanogen  compounds.  It  contains  albuminates,  which  are 
thrown  down  as  a  voluminous  white  precipitate  by  tannic  acid,  and 


362  PROTECTIVE    INOCULATIONS. 

are  redissolved  by  hot  water  containing  sodium  chloride  and  very 
dilute  potash  solution.  The  elementary  analysis  gave  N  5.90  per 
cent,  C  35.19  per  cent,  and  H  7.02  per  cent.  The  results  obtained 
are  believed  to  show  that  the  active  substance  present  in  the  lymph 
is  a  toxalbumin.  In  experiments  made  with  Koch's  lymph  in  Pas- 
teur's laboratory  by  Bardach,  a  very  decided  elevation  of  temperature 
was  produced  in  tuberculous  guinea-pigs  by  the  subcutaneous  injection 
of  0.1  gramme,  and  a  fatal  result  by  the  injection  of  0.2  to  0.5  gramme. 
In  man  a  decided  febrile  reaction  is  produced  in  tuberculous  patients 
by  very  much  smaller  doses — 0.001  cubic  centimetre. 

Hammerschlag,  in  his  chemical  researches,  found  that  the  tubercle 
bacillus  yields  a  larger  proportion  of  substances  soluble  in  alcohol  and 
ether  than  any  other  bacilli  tested  (twenty-seven  per  cent).  The  alco- 
holic extract  contains  fat,  lecithin,  and  a  toxic  substance  which  pro- 
duces convulsions  in  rabbits  and  guinea-pigs.  The  portion  insoluble 
in  alcohol  and  ether  contains  cellulose  and  an  albuminoid  substance. 
No  ptomains  were  found,  but  a  toxalbumin  was  isolated,  which 
caused  an  elevation  of  temperature  in  rabbits  of  1°  to  2°  C.,  lasting 
for  a  day  or  two. 

Koch  (1891)  has  given  a  full  account  of  his  method  of  prepari-ng 
crude  tuberculin,  and  also  the  process  by  which  he  obtains  from  this 
a  tuberculin  which  appears  to  be  pure,  or  nearly  so.  To  obtain  con- 
siderable quantities  of  the  crude  product  the  tubercle  bacillus  is  culti- 
vated in  an  infusion  of  calves'  flesh,  or  of  beef  extract  to  which  one 
per  cent  of  peptone  and  four  or  five  per  cent  of  glycerin  have  been 
added.  This  culture  liquid  must  be  made  slightly  alkaline,  and  it 
is  placed  in  flasks  with  a  flat  bottom,  which  should  not  be  more  than 
half  filled — thirty  to  fifty  cubic  centimetres.  The  inoculation  is  made 
upon  the  surface  with  small  masses  from  a  culture  upon  blood  serum 
or  glycerin  agar.  By  accident  Koch  discovered  that  these  masses 
floating  upon  the  surface  give  rise  to  an  abundant  development,  and 
to  the  formation  of  a  tolerably  thick  and  dry  white  layer,  which  finally 
covers  the  entire  surface.  At  the  end  of  six  to  eight  weeks  develop- 
ment ceases,  and  the  layer  after  a  time  sinks  to  the  bottom,  breaking 
up  meanwhile  into  fragments.  These  cultures,  after  their  purity  has 
been  tested  by  a  microscopical  examination,  are  poured  into  a  suitable 
vessel  and  evaporated  to  one- tenth  the  original  volume  over  a  water 
bath.  The  liquid  is  then  filtered  through  procelain.  The  crude  tu- 
berculin obtained  by  this  process  contains  from  forty  to  fifty  per  cent 
of  glycerin,  and  consequently  is  not  a  suitable  medium  for  the  develop- 
ment of  saprophy tic  bacteria,  if  they  should  by  accident  be  introduced 
into  it.  It  keeps  well  and  preserves  its  activity  indefinitely. 


PROTECTIVE    INOCULATIONS.  3G3 

From  this  crude  tuberculin  Koch  has  obtained  a  white  precipitate 
with  sixty-per-cent  alcohol  which  has  the  active  properties  of  the 
crude  tuberculin  as  originally  prepared.  This  is  fatal  to  tuberculous 
guinea-pigs  in  doses  of  two  to  ten  milligrammes.  It  is  soluble  in 
water  and  in  glycerin,  and  has  the  chemical  reactions  of  an  albuminous 
body.  In  preparing  it  one  volume  and  a  half  of  absolute  alcohol  is 
added  to  one  volume  of  the  crude  tuberculin,  and,  after  stirring  it  to 
secure  uniform  admixture,  this  is  put  aside  for  twenty -four  hours. 
At  the  end  of  this  time  a  flocculent  deposit  will  be  seen  at  the  bot- 
tom of  the  vessel.  The  fluid  above  this  is  carefully  poured  off;  and 
an  equal  quantity  of  sixty-per-cent  alcohol  is  poured  into  the  vessel 
for  the  purpose  of  washing  the  precipitate.  This  is  again  allowed  to 
settle,  and  the  procedure  is  repeated  three  or  four  times,  after  which 
the  precipitate  is  washed  with  absolute  alcohol.  It  is  then  placed 
upon  a  filter  and  dried  in  a  vacuum  exsiccator. 

The  "  tuberculocidin  "  of  Klebs  is  a  purified  tuberculin  obtained  by 
precipitation  with  alcohol.  The  precipitate  is  washed  in  chloroform 
and  then  dissolved  in  a  mixture  of  carbolic  acid  and  glycerin. 

Bujwid  (1894)  prepares  tuberculin  as  follows :  He  uses  cultures  on 
glycerin  agar  or  in  glycerin  bouillon  which  have  been  kept  at  a  suit- 
able temperature  for  five  to  eight  weeks.  The  glycerin-agar  cultures 
are  treated  with  distilled  water  by  which  the  tuberculin  is  extracted. 
After  adding  the  water  the  test  tubes  are  kept  in  a  cool  place  for 
twenty -four  hours,  and  this  is  repeated  two  or  three  times.  The  ex- 
tract from  the  agar  cultures  or  the  bouillon  cultures  is  then  sterilized 
by  exposure  for  from  five  to  ten  minutes  to  a  temperature  of  100°  C. ; 
then  filtered  through  a  Chamberland  filter ;  then  evaporated  at  a  low 
temperature  to  a  syrup-like  consistence.  When  this  crude  tuberculin 
is  dropped  into  ten  times  its  volume  of  strong  alcohol  a  brown  pre- 
cipitate is  thrown  down  which  contains  the  active  principle.  From 
the  tubercle  bacilli  obtained  by  filtering  his  cultures  Bujwid  also 
obtained  an  active  substance  which  in  doses  of  two  milligrammes 
caused  an  elevation  of  2°  C.  in  the  temperature  of  an  infected  guinea- 
pig.  This  substance  was  obtained  by  digesting  the  bacilli  for  two 
months  in  glycerin  and  water  (three  per  cent  of  glycerin),  filtering 
and  evaporating  the  extract,  and  precipitation  in  six  volumes  of 
ninety-five-per-cent  alcohol.  The  precipitate  when  dried  was  in  the 
form  of  a  white  powder. 

Helm  an  (1894)  obtains  tuberculin  from  potato  cultures.  The  sec- 
tions of  potato  are  neutralized  by  leaving  them  for  half  an  hour  in  a 
solution  of  one-half  to  one  per  cent  of  bicarbonate  of  soda,  after  which 
they  are  sterilized  for  twenty  minutes  in  the  autoclave  at  120°  C.  The 


364  PROTECTIVE    INOCULATIONS. 

best  results  were  obtained  when  the  potatoes  were  wet  with  a  five-per- 
cent solution  of  glycerin.  The  sections  of  potato  were  placed  in  Petri's 
dishes  upon  blotting  paper  wet  with  a  sublimate  solution,  and  the 
dishes  containing  the  cultures  were  surrounded  with  cotton  wet  with 
the  same  solution.  The  cultures  were  subsequently  treated  with  dis- 
tilled water,  to  extract  the  active  principle,  which  was  also  obtained 
from  the  bacilli  by  mixing  them  with  glycerin  in  the  proportion  of 
1:10. 

Numerous  experiments  have  been  made  with  dead  tubercle  bacilli, 
as  well  as  with  the  toxic  products  developed  in  cultures.  Hericourt 
and  Bichet  (1890)  found  by  experiment  that  old  cultures  heated  to  80° 
C.,  several  days  in  succession,  when  injected  into  a  vein  in  rabbits,  in 
the  dose  of  ten  to  twenty  cubic  centimetres,  caused  the  death  of  these 
animals.  Smaller  doses  from  which  the  animals  recovered  seemed  to 
make  them  less  susceptible  to  infection  than  control  animals,  but  the 
number  of  experiments  was  too  limited  to  establish  this  as  a  fact.  In 
a  subsequent  (1891)  communication  the  authors  named  claim  to  have 
succeeded  in  immunizing  rabbits  by  injecting  filtered  and  sterilized 
cultures  of  the  tubercle  bacillus,  either  subcutaneously  (five  to  fifteen 
cubic  centimetres)  or  into  a  vein  (twenty  to  forty  drops) .  The  injections 
were  repeated  every  second  or  third  day  for  a  period  of  fifteen  days, 
after  which  the  test  inoculation  was  made  with  a  culture,  obtained 
from  a  tuberculous  cow  in  one  series,  and  from  tuberculous  fowls  in 
another.  Four  vaccinated  rabbits  in  the  first  series  escaped  general 
tuberculosis,  while  four  out  of  eight  control  animals  died  tuberculous. 
In  the  second  series  five  vaccinated  animals  resisted  infection  and 
three  out  of  four  control  animals  died  tuberculous. 

De  Schweinitz  (1894)  has  reported  the  results  of  experiments  with 
attenuated  cultures  of  the  tubercle  bacillus,  and  has,  apparently,  suc- 
ceeded in  conferring  immunity  upon  guinea-pigs  by  inoculations 
with  such  cultures. 

Klebs  (1891) ,  in  experiments  on  guinea-pigs  and  rabbits,  convinced 
himself  that  the  fatal  result  of  an  inoculation  with  tubercle  bacilli  (in 
the  cavity  of  the  abdomen  or  subcutaneously  in  guinea-pigs,  and  in 
the  eye  in  rabbits)  was  greatly  delayed  by  injections  of  Koch's  tuber- 
culin (0.3  to  0.5  cubic  centimetre)  either  before  or  after  infection. 

Baumgarten  (1891),  in  experiments  upon  rabbits  inoculated  with 
tubercle  bacilli  in  the  anterior  chamber  of  the  eye,  failed  to  obtain 
favorable  results  from  treatment  with  Koch's  tuberculin  given  in  con- 
siderable doses  (0.5  to  one  gramme)  either  before  or  after  infection. 

The  results  reported  in  the  same  year  by  Gramatschikoff,  by 
Popoff,  by  Alexander,  and  by  Gasparini  and  Mercanti,  were  also  un- 


PROTECTIVE   INOCULATIONS.  365 

favorable  as  regards  au  immunizing  or  curative  effect  from  inocula- 
tions of  tuberculin  in  rabbits.  Donitz,  on  the  contrary,  arrives  at  the 
conclusion  that  when  early  treatment  is  instituted  iris  tuberculosis 
may  be  arrested  and  cured,  and  the  more  recent  experiments  of  Tru- 
cleau  (1893)  give  support  to  this  conclusion.  Baumgarten,  however, 
insists  that  the  tuberculin  treatment  does  not  prevent  metastasis  to 
the  lungs  after  inoculations  in  the  anterior  chamber  of  the  eye. 

Pfuhl  (1891)  treated  forty-seven  infected  guinea-pigs,  and  at  the 
date  of  his  report  forty-four  had  died  tuberculous,  but  the  date  of 
death  was  somewhat  postponed  by  the  treatment.  The  animals  not 
treated  succumbed  at  the  end  of  eight  weeks  (average  of  all  controls), 
and  those  treated  with  small  doses  of  tuberculin  lived,  on  the  average, 
ten  weeks.  With  larger  doses  still  more  favorable  results  were  ob- 
tained— four  lived  on  an  average  twelve  weeks,  and  three  were  still 
living,  eleven,  fifteen,  and  sixteen  weeks  after  infection,  at  the  date  of 
publication. 

Kitasato  (1892)  also  obtained  favorable  results  in  the  treatment 
of  infected  guinea-pigs,  and  arrives  at  the  conclusion  that  guinea-pigs 
which  have  been  cured  by  the  treatment  are  not  susceptible  to  a  sec- 
ond infection,  for  a  certain  time  at  least. 

Bujwid  (1892),  in  experiments  upon  guinea-pigs,  found  that  in- 
fected animals  which  received  from  0.05  to  0.1  gramme  of  tuberculin 
within  three  hours  showed  an  elevation  of  temperature  of  1.5°  to  2°  C. 
Thirteen  infected  guinea-pigs  treated  with  tuberculin  lived  from  two 
and  a  half  to  eight  months,  while  all  of  the  control  animals  (eighteen) 
died  in  from  six  to  nine  weeks.  The  animal  which  survived  eight 
months  was  found  not  to  be  tuberculous,  but  presented  evidence  of  re- 
covery from  a  former  tuberculous  process.  In  two  rabbits  inoculated 
in  the  anterior  chamber  the  iris  tuberculosis  was  favorably  influenced 
by  the  tuberculin  treatment,  but  general  infection  occurred,  and  the 
animals  died  about  the  same  time  as  the  controls.  Three  apes  were 
treated  without  any  apparent  result;  they  all  died  within  two  months 
after  infection. 

The  experiments  of  Gramatschikoff,  Czaplewski,  and  Roloff,  and 
of  Yamagiva,  published  in  1892,  show  that  the  tuberculin  treatment 
does  not  cure  tuberculous  infection  in  inoculated  guinea-pigs  and  rab- 
bits, and  that  the  bacilli  retain  their  vitality  in  such  animals  in  spite 
of  the  most  persistent  treatment. 

Hericourt  and  Eichet  (1892),  in  experiments  made  for  the  purpose 
of  immunizing  animals  against  tuberculous  infection,  failed  to  obtain 
positive  results  in  the  most  susceptible  species — guinea-pigs,  rabbits, 
and  apes — but  claim  to  have  succeeded  in  immunizing  dogs  by  intra- 


366  PROTECTIVE   INOCULATIONS. 

venous  injections  of  cultures  of  the  bacillus  of  tuberculosis  in  fowls. 
Animals  which  had  been  so  treated  after  an  interval  of  two  to  six 
months  received  an  intravenous  injection  of  one  cubic  centimetre  of  a 
culture  of  the  bacillus  tuberculosis  from  man.  This  was  fatal  to 
"  non-vaccinated  "  dogs,  as  a  rule,  in  about  three  weeks,  but  the  "  vac- 
cinated "  animals  survived  the  injection. 

The  results  obtained  by  Trudeau  (1893)  are  of  such  interest  that 
we  shall  quote  in  extenso  what  he  says  with  reference  to  preventive 
inoculations : 

"  Antitubercular  inoculation  was  first  tried  by  Falk  in  1883,  and  all 
attempts  in  this  direction  have  resulted  until  recently  in  but  uu  unbroken 
record  of  failures.  In  1890  I  added  my  name  to  the  list  of  those  who  found 
it  impossible  to  produce  immunity  in  animals  by  this  method.  In  1S1K), 
Martin  and  Grancher,  and  Courmont  and  Dor,  claimed  to  have  produced  in 
rabbits  a  certain  degree  of  immunity  by  previous  inoculation,  after  Pasteur's 
hydrophobia  method,  of  aviaii  tubercle  bacilli  of  graded  and  increasing  viru- 
lence. These  vaccinations  were,  however,  frequently  fatal  to  the  animals, 
and  the  immunity  obtained  was  but  slight.  Richet  and  He'ricourt  have  since 
claimed  to  produce  complete  immunity  in  dogs  by  intravenous  inoculations 
of  bird  tubercle  bacilli.  These  experimenters  found  that  though  harmless  to 
the  dog  when  first  derived  from  the  chicken,  bird  bacilli,  by  long  cultivation 
in  liquid  media,  become  pathogenic  for  this  animal,  and  by  thus  grading  the 
virulence  of  the  injections  complete  immunity  against  any  form  of  tubercu- 
lar infection  was  produced  in  the  dog.  As  yet  these  striking  results  have  not 
been  confirmed.  The  animals  which  I  now  present  to  you  illustrate  an  at- 
tempt I  have  made  along  the  same  line  to  produce  immunity  in  the  rabbit. 
Cultures  grown  directly  from  the  chicken's  lesions  in  bouillon  for,  first,  five 
weeks,  then  six  months,  were  twice  injected  subcutaneously  at  intervals  of 
twenty-one  days  in  doses  of  0.025  and  005,  and  a  third  injection  of  a  still 
older  culture  was  occasionally  given.  About  one  in  four  of  the  rabbits  died 
within  three  months,  profoundly  emaciated,  but  without  any  visible  tubercu- 
lar lesions.  The  remaining  animals  recovered  aud  were  apparently  in  good 
health,  when,  together  with  an  equal  number  of  controls,  they  were  inocu- 
lated in  the  anterior  chamber  of  the  eye  with  cultures  of  Koch's  bacillus 
derived  from  the  tuberculous  lesions  of  the  rabbit,  and  cultivated  about  three 
months  on  glycerin- agar.  The  results  of  these  inoculations  present  many 
points  of  interest.  In  the  controls,  as  is  usually  the  case,  if  the  operation 
has  been  done  carefully  and  aseptically,  and  with  a  moderate  amount  of 
dilute  virus,  two  days  after  the  introduction  of  the  virulent  material  in  the 
eye  little  or  no  irritation  is  observed,  and  little  is  to  be  noticed  for  two  weeks, 
when  a  steadily  increasing  vascularity  manifests  itself,  small  tubercles  ap- 
pear on  the  iris,  which  gradually  coalesce  and  become  cheesy,  intense  iritis 
and  general  inflammation  of  the  structures  of  the  eye  develop,  the  inocula- 
tion wound  becomes  cheesy,  and  in  six  to  eight  weeks  the  eye  is  more  or  less 
completely  destroyed  and  the  inflammation  begins  to  subside.  The  disease, 
however,  remains  generally  localized  in  the  eye  for  many  months,  and  even 
permanently.  In  the  vaccinated  animals,  on  the  contrary,  the  introduction 
of  the  virulent  bacilli  at  once  gives  rise  to  a  marked  degree  of  irritation.  On 
the  second  day  the  vessels  of  the  conjunctiva  are  tortuous  and  enlarged, 
whitish  specks  of  fibrinous-looking  exudation  appear  in  the  iris  and  in  the 
anterior  chamber,  and  more  or  less  intense  iritis  supervenes ;  but  at  the  end 
of  the  second  to  the  third  week,  when  the  eyes  of  the  controls  begin  to  show 
progressive  and  steadily  increasing  evidence  of  inflammatory  reaction,  the 
irritation  in  those  of  the  vaccinated  animals  begins  slowly  to  subside  and  the 
eyes  to  mend.  The  vascularity  is  less,  the  whitish  spots  of  fibriiious  material 


PROTECTIVE   INOCULATIONS.  367 

appear  smaller,  the  structures  of  the  eye  become  clearer,  the  inoculation 
wound  is  but  a  bluish  fibrous  scar,  until  in  from  six  to  twelve  weeks,  in  suc- 
cessful cases,  all  irritation  has  disappeared  and  the  eyes  present,  as  in  the 
animals  I  now  show  you,  but  fibrous  evidence  of  the  traumatism  and  the  in- 
flammatory processes  which  have  been  set  up  by  the  inoculation.  In  all  the 
controls,  as  you  see,  the  inoculation  wound  is  cheesy  and  the  cornea  and  iris 
are  more  or  less  destroyed  by  tubercle  and  cheesy  areas. 

"Some  of  the  protected  animals  slowly  relapse,  and  the  one  I  now  show 
you  has  small  tubercles  growing-  on  the  iris;  but  even  in  such  eyes  the 
entire  absence  of  caseation  is  noticeable,  and  the  disease  progresses  almost 
imperceptibly.  I  have  repeated  this  experiment  on  three  sets  of  rabbits  with 
about  the  same  results  each  time.  The  vaccinations  as  practised  are  of  them- 
selves, in  some  instances,  fatal :  but  the  fact  remains  that  where  recovery 
takes  place  a  marked  degree  of  immunity  has  been  acquired.  I  do  not  lay 
any  claim,  therefore,  to  have  produced  a  complete  or  permanent  immunity 
by  a  safe  method,  but  it  seems  to  me  that  these  eyes  constitute  a  scientific 
demonstration  of  the  fact  that  in  rabbits  preventive  inoculation  of  bird- 
tubercle  bacilli  can  retard,  and  even  abort,  an  otherwise  progressive  localized 
tubercular  process  so  completely  as  to  prevent  destruction  of  the  tissues 
threatened,  and  that  the  future  study  of  anti-tubercular  inoculation  may  not 
be  as  entirely  hopeless  as  it  has  until  recently  appeared." 

TYPHOID   FEVER. 

Brieger  (1885)  found  in  cultures  of  the  typhoid  bacillus  small 
amounts  of  volatile  fat  acids,  and  when  grape  sugar  has  been  added 
to  the  culture  medium  lactic  acid.  He  also  obtained  a  highly  alka- 
line basic  substance  possessing  toxic  properties  which  he  named 
typhotoxin  (C7H17NO2).  This  he  supposes  to  be  the  specific  product 
to  which  the  pathogenic  action  of  the  bacillus  is  due.  It  produces  in 
mice  and  guinea-pigs  salivation,  paralysis,  dilated  pupils,  diarrhoea, 
and  death. 

More  recent  experiments  by  Pfeiffer  (1894)  lead  him  to  conclude 
that  the  specific  poison  of  the  typhoid  bacillus  is  not  present  in  fil- 
tered cultures,  but  is  closely  associated  with  the  bacterial  cells.  Ac- 
cording to  Pfeiffer  the  bacillus  may  be  killed  by  a  temperature  of  54°  C. 
without  injury  to  this  toxic  substance.  The  fatal  dose  of  the  dead 
bacilli  is  from  three  to  four  milligrammes  per  one  hundred  grammes 
of  body  weight  for  guinea-pigs.  Susceptible  animals  may  be  im- 
munized by  means  of  this  toxic  substance,  and  their  blood  is  found  to 
contain  an  antitoxin  which  has  a  specific  bactericidal  action  upon  the 
typhoid  bacillus.  But,  according  to  Pfeiffer,  the  blood  serum  of  ani- 
mals immunized  in  this  way  does  not  differ  from  normal  serum  in 
its  action  on  bacillus  coli  communis  and  other  species  of  bacteria. 
These  results  are  believed,  by  the  author  referred  to,  to  settle  the 
question  of  the  specific  character  of  the  typhoid  bacillus,  and  to  dif- 
ferentiate it  from  nearly  allied  species.  The  presence  of  a  typhoid 
antitoxin  in  the  blood  serum  of  individuals  who  have  recently  suffered 
an  attack  of  typhoid  fever  has  also  been  demonstrated  by  Pfeiffer. 


368  PROTECTIVE   INOCULATIONS. 

Chantemesse  and  Widal  (1888)  first  showed  by  experiment  thai 
susceptible  animals  could  be  made  immune  against  the  pathogenic 
action  of  this  bacillus  by  the  subcutaneous  injection  of  sterilized  cul- 
tures. Having  found  that  four  drops  of  a  bouillon  culture,  three 
days  old,  injected  into  the  peritoneal  cavity  of  white  mice  caused  the 
death  of  these  animals  within  thirty-six  hours,  they  proceeded  to  in- 
ject small  quantities  (one-half  cubic  centimetre)  of  a  culture  which 
had  been  sterilized  by  heat,  and  found  that  after  several  such  protec- 
tive inoculations  the  mice  no  longer  succumbed  to  infection  by  an 
unsterilized  culture. 

In  experiments  made  upon  rabbits,  Bitter  (1892)  arrived  at  the 
conclusion  that  the  immunity  which  he  produced  in  these  animals  by 
the  intravenous  injection  of  concentrated  sterilized  (by  filtration)  cul- 
tures was  due  to  the  presence  of  an  antitoxin  in  the  blood  of  the 
immune  animals.  Having  found  that  control  animals  were  killed  by 
intravenous  injections  of  one  cubic  centimetre  of  his  concentrated 
solution  of  the  products  of  the  typhoid  bacillus,  he  added  to  twice 
this  amount  of  the  toxic  solution  a  certain  quantity  (?)  of  blood 
serum  from  an  immune  rabbit,  and  injected  the  mixture  into  the 
circulation  of  rabbits  with  a  negative  result.  Control  experiments 
in  which  the  toxic  solution  was  mixed  with  blood  serum  from  non- 
immune  animals  showed  that  this  had  no  antitoxic  effect,  and  the  ani- 
mals died.  Bruschettini  obtained  (1892)  similar  results  in  his  ex- 
periments upon  rabbits  with  cultures  sterilized  by  heat  (60°  C.).  He 
concludes  from  his  experiments  that  the  blood  serum  of  rabbits  im- 
munized in  this  way  not  only  possesses  antitoxic  properties,  but  that 
it  has  greater  germicidal  potency  for  the  typhoid  bacillus  than  the 
blood  of  normal  rabbits. 

Stern  (1892)  has  made  experiments  to  determine  whether  the  blood 
of  recent  convalescents  from  typhoid  has  greater  germicidal  power 
for  the  typhoid  bacillus  than  that  of  other  individuals.  The  result 
showed  that  the  blood  serum  from  persons  who  had  recently  recovered 
from  typhoid  fever  had  no  increased  germicidal  power,  but  rather 
showed  diminished  potency  for  the  destruction  of  typhoid  bacilli. 
But  blood  from  a  man  who  had  suffered  an  attack  seventeen  and  a 
half  years  previously  was  found  to  have  unusual  bactericidal  power, 
although  it  did  not  protect  white  mice  from  typhoid  infection.  On 
the  other  hand,  blood  from  recent  convalescents  served  to  immunize 
white  mice,  thus  indicating  the  presence  of  an  antitoxin.  This  is 
also  shown  by  the  experiments  of  Chantemesse  and  Widal  (1892), 
who  report  their  success  in  immunizing  susceptible  animals  by  in- 
jecting the  blood  serum  of  other  animals  previously  made  immune  by 


PROTECTIVE   INOCULATIONS.  369 

repeated  injections  of  sterilized  (by  heat)  cultures.  The  authors  last 
named  have  also  tested  the  blood  serum  of  typhoid-fever  patients,  of 
recent  convalescents  from  the  disease,  and  of  persons  who  had  suf- 
fered an  attack  some  years  before  the  experiment  was  made.  The  ex- 
periments were  made  upon  guinea-pigs.  The  authors  conclude  that 
"  in  general  the  guinea-pig  is  immunized  against  the  action  of  virulent 
typhoid  cultures  by  the  subcutaneous  injection  of  a  small  quantity 
of  serum  of  persons  who  have  suffered  an  attack  of  the  disease,  no 
matter  how  remote."  But  this  immunity  was  shown  to  be  of  short 
duration,  and  quite  different  from  that  induced  by  the  injection  of 
sterilized  cultures,  which  does  not  immediately  follow  the  introduc- 
tion of  the  toxic  substances,  but  requires  a  certain  number  of  days  for 
its  development.  The  degree  of  immunity  is  said  by  the  authors  last 
named  to  depend  to  a  considerable  extent  upon  the  dose  given,  and 
the  animals  treated  in  this  way  still  resisted  virulent  cultures  at  the 
end  of  two  months.  On  the  other  hand,  injections  of  blood  serum 
from  immune  individuals  were  effective  in  doses  of  a  single  cubic  cen- 
timetre, within  a  few  hours,  and  the  immunity  conferred  had  a  com- 
paratively brief  duration. 

Protective  inoculations  in  man  have  been  practised  on  quite  a  large 
scale  by  surgeons  of  the  English  army  in  India  and  in  South  Africa. 
The  method  of  Wright  has  been  followed  in  preparing  sterile  cultures 
for  inoculation.  Cultures  in  bouillon  are  made  and  kept  in  the  incu- 
bator at  37°  C.  for  two  or  three  weeks.  The  cultures  are  then  drawn 
into  small  glass  tubes,  which  are  sealed  by  heat.  The  tubes  are 
placed  in  a  vessel  containing  cold  water,  and  the  temperature  is  grad- 
ually raised  to  60°  C.,  where  it  is  maintained  for  five  minutes.  Plant- 
ings in  a  culture  medium  are  made  from  these  tubes  to  make  sure  that 
sterilization  is  complete.  As  a  further  protection  against  the  intro- 
duction of  living  bacteria,  one-half  per  cent  of  lysol  may  be  added  to 
the  sterilized  culture.  The  amount  used  for  protective  inoculations 
in  man  has  been  fixed  at  two-fifths  of  the  minimum  amount,  which 
would  be  fatal  to  a  guinea-pig  weighing  two  hundred  and  fifty 
grammes.  The  inoculation  gives  rise  to  a  well-marked  local  reaction, 
which  does  not  result  in  suppuration,  and  to  more  or  less  pronounced 
general  disturbance.  Usually  this  is  slight,  but  sometimes  rigors, 
nausea,  and  a  tendency  to  syncope  occur.  That  these  inoculations 
are  not  without  effect  is  shown  by  the  fact  that  the  blood  serum  of 
an  inoculated  individual  exercises  a  marked  agglutinating  action 
upon  the  typhoid  bacillus  in  a  recent  culture  (Widal  reaction).  This 
is  said  to  be  equal  to  that  resulting  from  an  attack  of  typhoid  fever. 

Cameron,  after  an  inoculation  practised  upon  himself,  found  that  at 
24 


370  PROTECTIVE   INOCULATIONS. 

the  end  of  twenty  days  his  blood  serum  exhibited  an  agglutinating 
power  forty  times  greater  than  that  of  normal  blood.  In  practice  it 
has  been  found  advisable  to  repeat  the  inoculation  at  the  end  of  a 
week.  Wright  reports  that  among  11,295  British  soldiers  inoculated 
in  India,  the  percentage  of  those  who  subsequently  contracted  typhoid 
fever  was  0.95,  while  2.5  per  cent  of  those  not  inoculated  suffered  an 
attack  of  this  disease.  According  to  Foulerton  the  soldiers  in  South 
Africa,  during  the  Boer  war,  who  have  been  inoculated  have  contracted 
typhoid  fever  in  the  proportion  of  six  per  thousand,  while  those  not 
inoculated  have  suffered  to  the  extent  of  nine  per  thousand.  How 
much  value  should  be  attached  to  these  statistics  it  is  difficult  to  say, 
on  account  of  the  numerous  factors  which  are  likely  to  influence  the 
result.  Thus  a  command  on  the  march  in  a  sparsely  inhabited  coun- 
try would  be  much  less  liable  to  suffer  from  typhoid  fever  than  another 
located  in  a  town  and  remaining  for  a  considerable  time  on  the  same 
camping  ground.  In  a  recent  report  (February,  1901)  Professor 
Wright  states  that  of  539  officers,  men,  and  women  connected  with  the 
Fifteenth  Hussars  at  Meerut,  India,  360  received  protective  inocula- 
tion in  England  against  typhoid  fever  and  179  did  not.  Of  the  former 
2  (0.55  per  cent)  were  admitted  to  the  hospital,  suffering  from  typhoid 
fever,  with  1  death  (0.27  per  cent) ;  while  of  the  latter  11  (6. 14  per  cent 
were  attacked  by  the  fever,  with  6  deaths  (3.35  per  cent). 

It  is  evident  that,  while  the  results  reported  are  encouraging,  this 
method  should  not  be  relied  upon  as  a  substitute  for  those  sanitary 
measures  which  must  be  our  main  reliance  for  the  prevention  of  epi- 
demics of  this  disease,  viz.,  sterilization  of  drinking-water,  disinfec- 
tion of  excreta,  sanitary  police  of  camps,  etc. 


V. 

PYOGENIC  BACTERIA. 

THE    demonstration  made  by   Ogston,   Rosenbach,  Passet,  and 
others  that  micrococci  are  constantly  present  in  the  pus  of  acute 
abscesses,  led  to  the  inference  that  there  can  be  no  pus  formation  in 
the  absence  of  microorganisms  of  this  class.     But  it  is  now  well 
established,  by  the  experiments  of  Grawitz,   De   Bary,  Steinhaus, 
Scheurlen,  Kaufmann,  and  others,  that  this  inference  was  a  mis- 
taken one,  and  that  certain  chemical  substances  introduced  beneath 
the  skin  give  rise  to  pus  formation  quite  independently  of  bacteria. 
Among  the  substances  tested  which  have  given  a  positive  result  are 
nitrate  of  silver,  oil  of  turpentine,  strong  liquor  ammoniae,   cada- 
verin,  etc.     The  demonstration  has  also  been  made  by  numerous  in- 
vestigators that  cultures  of  pus  cocci,  when  sterilized  by  heat,  still 
give  rise  to  pus  formation  when  injected  subcutaneously.     This  was 
first  established  by  Pasteur  in  1878,  who  found  that  sterilized  cul- 
tures of  his  "  microbe  generateur  du  pus "  induced  suppuration  as 
well  as  cultures  containing  the  living  microbe.     This  fact  lias  since 
been  confirmed,  as  regards  the  pus  staphylococci  and  various  bacilli, 
by  a  number  of  bacteriologists.     Wyssokowitsch  produced  abscesses 
containing  sterile  pus  by  injecting  subcutaneously  agar  cultures  of 
the  anthrax  bacillus  sterilized  by  heat.     Buchner  obtained  similar 
results  in  a  series  of  forty  experiments  from  the  injection  of  steril- 
ized cultures  of  Friedlander's  bacillus  ("  pneumococcus "),  and  has 
shown  that  the  pus-forming  property  belongs  to  the  bacterial  cells 
and  not  to  a  soluble  chemical  substance  produced  by  them.     When 
cultures  were  filtered  by  means  of  a  Chamberlain  filter  the  clear 
fluid  which  passed  through  the  porous  porcelain  was  without  effect, 
while  the  dead  bacteria  retained  by  the  filter  produced  aseptic  pus 
infiltration  in  the  subcutaneous    tissues  within  forty-eight    hours 
after  having  been  injected.     Subsequent  experiments  gave  similar 
results  with  seventeen  different  species  tested,  including  Staphylo- 
coccus  pyogenes  aureus,  Staphylococcus  cereus  flavus,  Sarcina  auran- 
tiaca,   Bacillus    prodigiosus,    Bacillus   Fitzianus,    Bacillus    subtilis, 
Bacillus  coli  communis,  Bacillus  acidi  lactici,  etc.     From  the  experi- 


372  PYOGENIC   BACTERIA. 

ments  made  to  determine  the  exact  cause  of  pus  formation  following 
the  injection  of  sterilized  cultures  Buchner  arrives  at  the  conclusion 
that  it  is  due  to  the  albuminous  contents  of  the  bacterial  cells. 

While  it  is  demonstrated  that  a  large  number  of  microorganisms, 
either  living  or  in  sterilized  cultures,  may  give  rise  to  the  formation 
of  pus,  the  extended  researches  of  Rosenbach,  Passet,  and  other 
bacteriologists  show  that  few  species  are  usually  concerned  in  the 
formation  of  acute  abscesses,  furuncles,  etc.,  in  man.  Of  these  the 
two  most  important,  by  reason  of  their  frequent  occurrence  and  path- 
ogenic power,  are  Staphylococcus  pyogenes  aureus  and  Strepto- 
coccus pyogenes ;  next  to  these  comes  Staphylococcus  pyogenes 
albus,  and  the  following  species  are  occasionally  found  :  Staphylo- 
coccus pyogenes  citreus,  Staphylococcus  cereus  flavus,  Staphylococcus 
cereus  albus,  Micrococcus  tenuis,  Bacillus  pyogenes  foetidus,  Micro- 
coccus  tetragenus,  Micrococcus  pneumonias  crouposae.  Two  or  more 
species  are  often  found  in  the  same  abscess  ;  thus  Passet,  in  thirty- 
three  cases  of  acute  abscess,  found  Staphylococcus  aureus  and  albus 
associated  in  eleven,  albus  alone  in  four,  albus  and  citreus  in  two, 
Streptococcus  pyogenes  alone  in  eight,  albus  and  streptococcus  in 
one,  and  albus,  citreus,  and  streptococcus  in  one.  Hoffa  found,  in 
twenty-two  cases  of  inguinal  bubo,  aureus  in  ten,  albus  in  nine,  and 
citreus  in  three.  Bumm,  in  ten  cases  of  puerperal  mastitis,  found 
aureus  in  seven  and  Streptococcus  pyogenes  in  three.  Rosenbach 
found  staphylococci  alone  sixteen  times,  Streptococcus  pyogenes  alone 
fifteen  times,  staphylococci  and  streptococci  associated  five  times, 
and  Micrococcus  tenuis  three  times  in  thirty-nine  acute  abscesses  and 
phlegmons  examined  by  him. 

Robb  and  Ghrisky  have  shown  that  under  the  most  rigid  antisep- 
tic treatment  microorganisms  are  constantly  found  attached  to  su- 
tures when  these  are  removed  from  wounds  made  by  the  surgeon, 
and  that  a  skin  abscess  frequently  results  from  the  presence  of  the 
most  common  of  these  microorganisms — Staphylococcus  epidermidis 
albus. 

The  authors  named  state  their  conclusions  as  follows  : 

"A  wound,  at  some  time  of  its  existence,  always  contains  organisms. 
They  occur  either  on  the  stitches  or  in  the  secretions. 

"  The  number  of  bacteria  is  influenced  by  the  constricting  action  of  the 
ligatures  or  drainage  tube,  or  anything  interfering  with  the  circulation  of 
the  tissues. 

' '  The  virulence  of  the  organisms  present  will  influence  the  progress  of 
the  wound. 

"The  body  temperature  is  invariably  elevated  if  the  bacteria  are  viru- 
lent; and,  indeed,  in  cases  where  many  of  the  less  virulent  organisms  are 
found,  almost  without  exception  there  is  some  rise  of  temperature. " 

The  organism  most  frequently  found — Staphylococcus  epidermi- 


PYOGENIC   BACTERIA.  373 

dis  albus — has  but  slight  virulence.  Out  of  forty-five  cases  in  which 
a  bacteriological  examination  was  made  this  micrococcus  was  ob- 
tained in  pure  cultures  in  thirty-three  ;  in  five  cases  it  was  associated 
with  Staphylococcus  pyogenes  aureus,  in  one  case  with  Streptococ- 
cus pyogenes,  in  three  cases  Streptococcus  pyogenes  was  obtained 
alone. 

In  abscesses  resulting  from  inflammation  of  the  middle  ear  the 
micrococcus  commonly  known  under  the  name  of  "  diplococcus 
pneumonise  "  —Micrococcus  pneumonias  crouposae — has  been  obtained 
in  pure  cultures  in  a  considerable  number  of  cases  when  the  pus  has 
been  examined  immediately  after  paracentesis  of  the  tympanic  mem- 
brane. We  shall  not,  however,  describe  this  among  the  pyogenic 
bacteria,  but  will  give  an  account  of  it  in  the  following  section  (Bac- 
teria in  Croupous  Pneumonia,  etc.).  Bacillus  pyocyaneus,  which  is 
described  by  some  authors  among  the  pyogenic  bacteria,  is  found 
only  in  the  pus  of  open  wounds,  where  its  presence  is  evidently  acci- 
dental. "We  shall  describe  it  among  the  chromogenic  saprophytes. 

STAPHYLOCOCCUS  PYOGENES  AUREUS. 

Synonym. — Micrococcus  of  infectious  osteomyelitis  (Becker). 

Observed  by  Ogston  (1881)  in  the  pus  of  acute  abscesses,  but  not 
differentiated  from  the  associated  staphylococci  and  the  streptococ- 
cus of  pus.  Obtained  by  Becker  from  the  pus  of  osteomyelitis  (1883). 
Isolated  from  the  pus  of  acute  abscesses  and  accurately  described  by 
Rosenbach  (1884)  and  by  Passet  (1885). 

The  Staphylococcus  pyogenes  aureus  is  a  facultative  parasite,  and 
is  the  most  common  pyogenic  micrococcus  found  in  suppurative  pro- 
cesses generally.  But  it  is  also  a  common  and  widely  distributed 
saprophyte,  which  finds  the  conditions  necessary  for  its  existence  on 
the  external  surface  of  the  human  body  and  of  moist  mucous  mem- 
branes. This  is  shown  by  the  researches  of  numerous  bacteriolo- 
gists. Thus  Ullmann  found  it  upon  the  skin  and  in  the  secretions  of 
the  mouth  of  healthy  persons,  and  also  in  the  dust  of  occupied  apart- 
ments, in  water,  etc. ;  Bockhart  obtained  it  in  cultures  from  the 
surface  of  the  body  and  from  the  dirt  beneath  the  finger  nails  of 
healthy  persons  ;  Biondi,  Yignal,  and  others  in  the  salivary  secre- 
tions ;  B.  Frankel  in  mucus  from  the  pharynx  ;  Von  Besser  and 
Wright  in  nasal  mucus  ;  Escherich  in  the  alvine  discharges  of 
healthy  infants  ;  C.  Frankel  in  the  air  ;  and  Lubbert  in  the  soil.  Its 
presence  in  the  air,  in  water,  or  in  the  soil  is,  however,  quite  excep- 
tional, and  is  probably  to  be  considered  the  result  of  accident,  its 
normal  habitat  as  a  saprophyte  appearing  to  be  rather,  .upon  the  sur- 
face of  the  body  and  of  mucous  membranes. 


374  PYOGENIC   BACTERIA. 

Morphology.—  Spherical  cells  having  a  diameter  of  0.7  ^  (Hade* 
lich)  to  0.9  /*  (0.87  /*  Passet),  solitary,  in  pairs,  or  in  irregular 
groups,  occasionally  in  chains  of  three  or  four  elements  or  in  groups 
of  four.  The  dimensions  vary  somewhat  in  dif- 
ferent  culture  media,  being  larger  in  a  favorable 
than  in  an  unfavorable  medium.  The  individual 
cells,  as  pointed  out  by  Hadelich,  consist  of  two 


hemispherical  portions  separated  from  each  other 
FIG.  79.-staphyiococ-  by  a  very  narrow  cleft,  which  is  not  visible  when 

cus    pyogenes     aureus,     ,-•  •>-,  j         ^  j      i_ 

from    a    drawing    by  tne  cells  are  deeply  stained,  but  may  be  demon- 
Rosenbach.  strated,  with  a  high  power,  by  staining  for  a  short 

time  (two  minutes  or  less)  in  a  solution  of  f  uchsin  in  aniline  water. 

This  micrococcus  stains  quickly  in  aqueous  solutions  of  the  basic 
aniline  colors,  and  may  also  be  stained  with  acid  carmine  and  hsema- 
toxylin.  It  is  not  decolorized  by  iodine  solution  when  stained  with 
methyl  violet  —  Gram's  method. 

Biological  Characters.  —  Staphylococcus  pyogenes  aureus  grows 
either  in  the  presence  or  absence  of  oxygen,  and  is  consequently  a 
facultative  anaerobic.  It  multiplies  rapidly  at  a  temperature  of  18° 
to  20°  C.  in  milk,  flesh  infusions,  and  various  other  liquid  media, 
and  in  nutrient  gelatin  or  agar.  It  liquefies  gelatin,  and  in  stab 
cultures  liquefaction  occurs  all  along  the  line  of  puncture,  forming  a 
pouch  which  is  largest  above  and  at  the  end  of  three  or  four  days  has 
extended  to  the  full  capacity  of  the  test  tube  at  the  surface.  The 
liquefied  gelatin  in  this  pouch  is  at  first  opaque  from  the  presence  of 
little  agglomerations  of  micrococci  in  suspension,  but  after  a  time 
these  are  deposited  and  the  gelatin  becomes  transparent.  During 
the  period  of  active  growth  the  cocci  accumulate  near  the  surface  of 
the  gelatin,  and,  in  contact  with  the  air,  the  characteristic  golden-yel- 
low pigment  is  produced.  By  the  subsidence  of  the  colored  masses 
of  cocci  from  this  superficial  stratum  a  yellow  deposit  is  gradually 
formed  at  the  bottom  of  the  pouch  of  liquefied  gelatin  (Fig.  80).  This 
pigment,  which  is  the  principal  character  distinguishing  the  micro- 
coccus  under  consideration  from  certain  other  liquefying  staphylo- 
cocci,  is  only  formed  in  the  presence  of  oxygen.  Upon  the  surface 
of  nutrient  agar  development  occurs  in  the  form  of  a  moist,  shining 
layer,  with  more  or  less  wavy  outlines,  having  at  first  a  pale-yellow 
color,  which  soon  deepens  to  an  orange-  or  golden-yellow.  The  col- 
onies which  develop  upon  agar  plates  are  spherical  and  opaque,  and 
usually  acquire  the  golden-yellow  color  within  a  few  days.  Colonies 
on  gelatin  plates  or  in  Esmarch  roll  tubes  first  appear  as  small  white 
dots,  which  later  are  more  or  less  granular  in  appearance  and  present 
the  yellow  color,  especially  towards  the  centre  ;  but,  owing  to  the 
extensive  liquefaction  of  the  gelatin  caused  by  them,  their  develop- 


PYOGENIC   BACTERIA. 


375 


ment  can  only  be  followed  for  two  or  three  days.  Upon  potato,  at  a 
temperature  of  35°  to  37°  C.,  a  rather  thick,  moist  layer  of  consider- 
able extent  forms  at  the  end  of  twenty-four  to  forty-eight  hours ; 
this  is  also  at  first  of  a  pale-yellow,  and  later 
of  an  orange-yellow  color.  The  temperature 
mentioned  is  most  favorable  for  the  rapid 
development  of  this  micrococcus,  although 
multiplication  may  occur  at  a  comparatively 
low  temperature  and  is  tolerably  abundant  at 
the  ordinary  room  temperature. 

Cultures  of  the  "golden  Staphylococcus," 
and  especially  those  upon  potato,  give  off  a 
peculiar  odor  which  resembles  that  of  sour 
paste.  When  cultivated  in  milk  it  gives  rise 
to  the  formation  of  lactic  and  butyric  acids 
and  to  coagulation  of  the  casein.  No  poison- 
ous ptomaines  or  toxalbumins  have  been  iso- 
lated from  cultures  of  this  micrococcus,  but, 
like  other  liquefying  bacteria,  it  forms  a  sol- 
uble peptonizing  ferment,  by  which  gelatin 
may  be  liquefied  independently  of  the  living 
microorganism.  While  the  Staphylococcus 
aureus  gives  rise  to  the  production  of  acids— 
principally  lactic  acid — in  media  containing  Staphylococcus  pyogenes  aureus 
glucose  or  lactose,  it  has  also  been  shown  by  ( 

Brieger  that  ammonia  is  one  of  the  products  of  its  vital  activity. 
Unlike  some  other  pathogenic  bacteria,  it  is  able  to  grow  in  a  medium 
having  a  distinctly  acid  reaction.  A  non-poisonous  basic  substance 
has  been  isolated  by  Brieger  from  old  cultures  in  meat  infusion  which 
differs  from  any  of  the  ptomaines  obtained  by  him  from  other  sources. 

The  thermal  death-point  of  this  micrococcus,  in  recent  cultures  in 
flesh-peptone-gelatin,  as  determined  by  the  writer,  is  between  56°  and 
58°  C. ,  the  time  of  exposure  being  ten  minutes.  When  "in  a  desic- 
cated condition  a  much  higher  temperature  is  required — 90°  to  100°  C. 
— for  its  destruction  ;  and  it  retains  its  vitality  for  more  than  ten 
days  when  dried  upon  a  cover  glass  (Passet).  It  retains  its  vitality 
for  a  long  time  in  cultures  in  nutrient  gelatin  or  agar,  and  may  grow 
when  transplanted  from  such  cultures  even  at  the  end  of  a  year. 

Very  numerous  experiments  have  been  made  to  determine  the 
proportion  of  various  chemical  agents  required  to  destroy  the  vitality 
or  to  restrain  the  growth  of  this  important  pyogenic  micrococcus. 
The  extended  researches  of  Lubbert  (1886)  with  reference  to  the 
antiseptic  power  of  agents  added  to  a  suitable  culture  medium — nu- 
trient gelatin — gave  the  following  results  :  Development  was  pre- 


FIG.    80.— Gelatin    culture   of 


376  PYOGENIC   BACTERIA. 

vented  by  the  agents  named  in  the  proportion  given  :  Nitric  acid, 
1  :  797  ;  phosphoric  acid,  1  :  750  ;  boracic  acid,  1  :  327  ;  oxalic  acid, 
1  : 433  ;  acetic  acid,  1  :  720  ;  citric  acid,  1  :  433  ;  lactic  acid,  1  : 350  ; 
benzoic  acid,  1  : 400 ;  salicylic  acid,  1 :  G55  ;  iodine  dissolved  with 
potassium  iodide,  1:1,100;  arsenite  of  potash,  1:733;  mercuric 
chloride,  1  :  81,400  ;  chloral  hydrate,  1  : 133  ;  carbolic  acid,  1  :  814  ; 
thymol,  1  : 11,000  ;  resorcin,  1  : 122  ;  hydrochinon,  1  :  353  ;  kairin, 
1  : 407  ;  antipyrin,  1  :  26  ;  muriate  of  quinine,  1  : 550  ;  muriate  of 
morphia,  1  :  60.  For  the  destruction  of  vitality  very  much  larger 
amounts  are  required.  In  Bolton's  experiments  (1887)  a  one-per-cent 
solution  of  carbolic  acid  was  successful  after  two  hours'  exposure, 
but  two  per  cent  failed  to  completely  destroy  vitality  in  the  same 
time  ;  one  per  cent  of  sulphate  of  copper  was  also  successful,  and  but 
a  single  colony  developed  after  exposure  to  a  solution  of  1  : 200.  In 
the  experiments  of  Gartner  and  Plagge  the  Staphylococcus  aureus  in 
bouillon  cultures  is  said  to  have  been  killed  in  a  few  seconds  (eight) 
by  a  solution  of  mercuric  chloride  of  the  proportion  of  1 : 1,000  ;  Behr- 
ing  found  it  was  killed  by  the  acid  sublimate  solution  of  La  Place, 
in  the  proportion  of  1  : 1,000,  in  ten  minutes ;  Tarnier  and  Vignal 
found  that  a  solution  of  1  : 1,000  was  successful  in  two  minutes. 
Abbott  (1891)  has  shown  that  in  the  same  culture  there  may  be  a 
considerable  difference  in  the  resisting  power  of  the  cocci,  and  that 
while  frequently  all  are  destroyed  in  five  minutes  by  a  1  : 1,000  solu- 
tion, it  occurs  quite  as  frequently  that  some  may  survive  after  an  ex- 
posure of  ten,  twenty,  and  even  thirty  minutes. 

Pathogenesis. — Subcutaneous  inoculation  with  a  small  quantity 
of  a  culture  of  Staphylococcus  pyogenes  aureus  is  without  result  in 
rabbits,  guinea-pigs,  or  mice,  but  when  a  considerable  quantity  is 
injected  beneath  the  skin  of  a  rabbit  or  a  guinea-pig  an  abscess  is 
produced,  which  usually  results  in  recovery,  but  may  give  rise  to 
general  infection  and  the  death  of  the  animal.  Injection  into  a 
vein  or  into  the  cavity  of  the  abdomen  in  the  animals  mentioned 
usually  induces  a  fatal  result  within  a  few  days.  The  most  charac- 
teristic pathological  changes  are  found  in  the  kidneys,  which  con- 
tain numerous  small  collections  of  pus  and  under  the  microscope 
present  the  appearances  resulting  from  embolic  nephritis.  Many  of 
the  capillaries  and  some  of  the  smaller  arteries  of  the  cortex  are 
plugged  up  with  thrombi  consisting  of  micrococci.  Metastatic  ab- 
scesses may  also  be  found  in  the  joints  and  muscles.  The  micro- 
cocci  may  be  recovered  in  pure  cultures  from  the  blood  and  the 
various  organs  ;  but  they  are  not  numerous  in  the  blood,  and  a  sim- 
ple microscopical  examination  will  often  fail  to  demonstrate  their 
presence. 

Animals  frequently  survive  the  injection  of  a  small  quantity  of 


PYOGENIC   BACTERIA.  377 

a  pure  culture  made  directly  into  the  circulation,  and  there  is  evi- 
dence that  the  pathogenic  potency  of  this  micrococcus  may  vary 
considerably  as  a  result  of  conditions  relating  to  its  origin  and  culti- 
vation in  the  animal  body  or  in  artificial  media.  When  injected  in 
considerable  quantities  it  may  be  obtained  in  cultures  from  the 
urine,  but  not  sooner  than  six  or  eight  hours  after  the  injection,  and 
not  until  the  formation  of  purulent  foci  in  the  kidneys  has  already 
occurred  ( Wy ssoko wit sch) . 

The  pyogenic  properties  of  this  micrococcus  have  been  demon- 
strated upon  man  by  the  experiments  of  Garre,  of  Bockhart,  and  of 
Bumm.  The  first-named  observer  inoculated  a  small  wound  at  the 
edge  of  one  of  his  finger  nails  with  a  minute  quantity  of  a  pure  cul- 
ture, and  a  subepidermal,  purulent  inflammation  extending  around 


;£)>       oZ^'dCT,'.»T-(     N-^-»rijr,i  -  f- 


FIG.  81.— Vertical  section  through  a  subcutaneous  abscess  caused  by  inoculation  with  staphylo- 
cocci,  in  the  rabbit,  forty  eight  hours  after  infection;  margin  towards  the  normal  tissue.  X  950. 
(Baumgarten.) 

the  margin  of  the  nail  resulted  from  the  inoculation.  Staphylococ- 
cus  aureus  was  recovered  in  cultures  from  the  pus  thus  formed.  A 
more  extensive  and  extremely  satisfactory  experiment  was  subse- 
quently made  by  Garre,  who  applied  a  considerable  quantity  of  a 
pure  culture  obtained  from  the  above-mentioned  source — third  gene' 
ration — to  the  uninjured  skin  of  his  left  forearm.  At  the  end  of 
four  days  a  large  carbuncle,  surrounded  by  isolated  furuncles,  de- 
veloped at  the  point  where  the  culture  had  been  applied.  This  ran 
the  usual  course,  and  it  was  several  weeks  before  it  had  completely 
healed.  No  less  than  seventeen  scars  remained  to  give  evidence  of 
the  success  of  the  experiment. 

In  Bockhart's  experiments  a  similar  but  milder  result  was  ob- 
tained,  the  conditions  having  been  somewhat  different.     A  small 


378  PYOGENIC   BACTERIA. 

quantity  of  an  agar  culture  was  suspended  in  0.5-per-cent  salt  solu- 
tion, and  this  was  rubbed  upon  the  uninjured  skin  of  the  left  fore- 
arm. By  gentle  scratching  with  a  disinfected  finger  nail  the  epithe- 
lium was  removed  in  places  over  the  area  to  which  the  micrococcus 
had  been  applied.  As  a  result  of  this  procedure  numerous  impe- 
tigo pustules  and  occasionally  a  genuine  furuncle  developed.  Por- 
tions of  the  skin  containing  the  smaller  pustules  were  excised  and 
examined  microscopically.  As  a  result  of  this  examination  Bock- 
hart  concluded  that  the  cocci  penetrate  by  way  of  the  hair  follicles, 
the  sebaceous  and  sudoriparous  glands,  or,  where  the  epidermis  had 
been  removed  by  scratching,  directly  to  the  deeper  layers  of  the  skin. 

In  Bumm's  experiments,  made  upon  himself  and  several  other 
persons,  Staphylococcus  aureus  suspended  in  sterilized  salt  solution 
was  injected  beneath  the  skin.  An  abscess  resulted  in  every  case. 

The  very  extended  researches  made  by  bacteriologists  during  the 
past  five  or  six  years  show  that  the  golden  Staphylococcus  is  the 
most  common  pyogenic  microorganism.  Its  presence  has  been  de- 
monstrated not  only  in  furuncles  and  carbuncles,  but  also  in  various 
pustular  affections  of  the  skin  and  mucous  membranes — impetigo, 
sycosis,  phlyctenular  conjunctivitis  ;  in  purulent  conjunctivitis  and 
inflammation  of  the  lacrymal  sac  ;  in  acute  abscesses  formed  in  the 
lymphatic  glands,  the  parotid  gland,  the  tonsils,  the  mammae,  etc. ; 
in  metastatic  abscesses  and  purulent  collections  in  the  joints  ;  in  em- 
pyema  ;  in  infectious  osteomyelitis  ;  and  in  ulcerative  endocarditis. 
The  evidence  relating  to  its  presence  and  etiological  import  in  the 
last-mentioned  affections  demands  special  consideration. 

Infectious  osteomyelitis  appears  from  the  researches  of  Becker, 
Rosenbach,  Krause,  Passet,  and  others,  to  be  usually  due  to  the  pre- 
sence of  Staphylococcus  aureus,  although  Kraske  has  shown  that  in 
certain  cases  this  is  associated  with  other  microorganisms.  Becker, 
who  obtained  this  micrococcus  from  the  pus  of  osteomyelitis  in  1883, 
was  the  first  to  show  by  experiment  that  the  same  affection  might  be 
induced  in  rabbits  by  injecting  cultures  of  the  micrococcus  into  the 
circulation,  after  having  crushed  or  fractured  a  bone  in  one  of  its 
legs.  The  animal  usually  died  in  from  twelve  to  fourteen  days  and 
presented  the  usual  appearances  of  osteomyelitis  at  the  fractured 
point.  The  abundant  yellowish-white  pus  contained  the  golden 
Staphylococcus  which  was  described  by  Becker,  and  subsequently 
known  in  the  bacteriological  laboratories  of  Germany  as  the  "  mi- 
crococcus of  infectious  osteomyelitis."  Becker's  experimental  re- 
sults have  been  confirmed  by  Krause  and  Rosenbach;  and  Rodet,  by 
injecting  smaller  quantities  of  a  culture  into  the  circulation,  has  suc- 
ceeded in  producing  an  osteomyelitis  without  previous  injury  to  the 
bone. 


PYOGENIC   BACTERIA.  379 

Ulcerative  endocarditis  has  been  shown  by  the  researches  of 
numerous  bacteriologists  to  be  occasionally  accompanied  by  a  mycotic 
invasion  of  the  affected  tissues  by  the  golden  staphylococcus ;  in 
other  cases  Streptococcus  pyogenes  is  present.  The  researches  of 
Weichselbaum,  and  of  E.  Frankel  and  Sanger,  also  show  that  it  is 
present  in  a  certain  proportion  of  the  cases,  at  least,  of  endocarditis 
verrucosa,  although  in  smaller  numbers.  That  the  diseased  condi- 
tion of  the  cardiac  valves  in  ulcerative  endocarditis  is  due  to  mycotic 
invasion  is  now  generally  admitted  and  is  supported  by  experimental 
evidence.  Rosenbach  first  (1873)  produced  an  endocarditis  in  lower 
animals  by  mechanical  injury  to  the  cardiac  valves,  effected  by  in- 
troducing a  sound  through  the  aorta.  Following  his  method,  Wys- 
sokowitsch  (1885),  after  injuring  the  cardiac  valves  in  rabbits,  in- 
jected into  the  circulation  pure  cultures  of  various  bacteria.  He 
obtained  positive  results  with  Staphylococcus  aureus  and  Strepto- 
coccus pyogenes  only.  When  these  micrococci  were  injected  into 
the  trachea  or  subcutaneously  the  result  was  negative,  as  was  the 
case  when  very  few  cocci  were  injected  into  a  vein,  or  when  two 
days  or  more  were  allowed  to  elapse  after  injury  to  the  cardiac 
valves.  Subsequently  Weichselbaum,  Prudden,  and  Frankel  and 
Sanger  obtained  confirmatory  results,  thus  establishing  the  fact  that 
when  the  valves  are  first  injured  mechanically  (or  chemically— 
Prudden)  the  injection  into  a  vein  of  a  pure  culture  of  Staphylococcus 
aureus  gives  rise  to  a  genuine  ulcerative  endocarditis.  It  has  been 
further  shown  by  Ribbert  that  the  same  result  may  be  obtained  with- 
out previous  injury  to  the  valves  by  injecting  into  a  vein  the  staphy- 
lococcus from  a  potato  culture  suspended  in  water.  In  his  experi- 
ments not  only  the  micrococci  from  the  surface  but  the  superficial 
layer  of  the  potato  was  scraped  off  with  a  sterilized  knife  and  mixed 
with  distilled  water  ;  and  the  successful  result  is  ascribed  to  the  fact 
that  the  little  agglomerations  of  micrococci  and  infected  fragments 
of  potato  attach  themselves  to  the  margins  of  the  valves  more  readily 
than  isolated  cocci  would  do.  In  these  experiments  the  mitral  and 
tricuspid  valves  were  affected,  while  the  semilunar  valves  remained 
intact.  In  ulcerative  endocarditis  it  is  evident  that  cocci  detached 
from  the  diseased  valves  must  find  their  way  into  the  circula- 
tion. As  a  matter  of  fact,  masses  of  micrococci  are  carried  away  by 
the  blood  stream  and  form  emboli  in  various  parts  of  the  body,  which 
become  secondary  foci  of  infection  and  give  rise  to  local  necrotic 
changes  and  accumulations  of  pus.  While  this  undoubtedly  occurs, 
it  is  generally  admitted  that  the  mycotic  infection  of  the  cardiac 
valves  is  usually  a  secondary  affection,  resulting  from  the  transpor- 
tation of  micrococci  in  the  blood  current  from  some  other  infected 
focus.  But  there  is  no  general  development  of  micrococci  in  the  cu> 


380  PYOGENIC   BACTERIA. 

dilating  fluid,  and  in  man,  as  in  animals  infected  experimentally,  a 
microscopic  examination  of  the  blood  for  microorganisms  usually 
gives  a  negative  result.  Culture  experiments  may,  however,  demon- 
strate their  presence.  Thus  recent  investigations  by  Netter,  Eisel- 
berg,  and  others  show  that  the  pus  cocci  are  usually  present  in  the 
blood  in  small  numbers,  as  demonstrated  by  culture  experiments,  in 
septic  infection  from  wounds. 

STAPHYLOCOCCUS   PYOGENES   ALBUS. 

Isolated  by  Rosenbach  (1884)  from  the  pus  of  acute  abscesses,  in 
which  it  is  sometimes  the  only  microorganism  present,  and  some- 
times associated  with  other  pus  cocci.  In  thirty-three  acute  abscesses 
examined  by  Passet  (1885)  it  was  associated  with  Staphylococcus 
aureus  in  eleven,  with  Staphylococcus  citreus  in  two,  with  Strepto- 
coccus pyogenes  in  one,  with  both  Staphylococcus  citreus  and  Strep- 
tococcus pyogenes  in  one,  and  was  obtained  alone  from  four. 

In  its  morphology  this  micrococcus  is  identical  with  the  preced- 
ing, but  it  is  distinguished  from  it  by  the  absence  of  pigment  and 
by  being  somewhat  less  pathogenic.  Surface  cultures  upon  nutrient 
agar  or  potato  have  a  milk-white  color.  It  liquefies  gelatin  in  the 
same  way  as  does  the  golden  Staphylococcus,  but  the  deposit  at  the 
bottom  of  the  liquefied  gelatin  is  without  color.  In  the  temperature 
conditions  favorable  to  its  growth,  and  in  its  biological  characters 
generally,  with  the  exceptions  noted,  it  is  not  to  be  distinguished 
from  the  species  previously  described.  According  to  Fliigge,  it  is 
more  common  than  aureus  among  many  of  the  lower  animals. 

Pathogenesis. — Fortunati  has  tested  the  comparative  pathogenic 
power  of  Staphylococcus  aureus  and  Staphylococcus  albus  by  inocu- 
lations into  the  cornea  of  rabbits.  A  purulent  infiltration  of  the 
cornea  and  panophthalmitis  resulted  when  Staphylococcus  aureus 
was  inoculated  upon  the  surface  of  the  cornea  by  scratching  with  an 
infected  needle,  but  inoculations  made  in  the  same  way  with  Staphy- 
lococcus albus  healed  spontaneously  or  gave  rise  to  a  perforating 
ulcer.  After  paracentesis  of  the  cornea  with  an  instrument  infected 
with  Staphylococcus  aureus  panophthalmitis  developed  in  thirty  hours; v 
the  same  result  occurred  at  the  end  of  sixty  to  seventy-two  hours 
when  the  instrument  was  infected  with  Staphylococcus  albus.  When 
a  sterilized  instrument  was  used  the  result  was  negative.  In  bacteri- 
ological researches  made  by  Gallenga,  in  cases  of  panophthalmitis  in 
man,  Staphylococcus  albus  was  found  in  ten  cultures  and  Staphy- 
lococcus aureus  in  nine. 

Staphylococcus  Epidermidis    Albus   (Welch). 
The  researches  of  Welch  show  that  a  white  Staphylococcus,  prob- 
ably identical  with  Staphylococcus  pyogenes  albus  of  Rosenbach,  is 


PYOGENIC   BACTERIA.  3$1 

the  most  common  microorganism  upon  the  surface  of  the  body,  and 
that  "  it  is  very  often  present  in  parts  of  the  epidermis  deeper  than 
can  be  reached  by  any  known  means  of  cutaneous  disinfection  save 
the  application  of  heat."  With  reference  to  this  coccus  Welch 
says : 

"So  far  as  our  observations  extend — and  already  they  amount  to  a 
large  number — this  coccus  may  be  regarded  as  a  nearly,  if  not  quite,  con- 
stant inhabitant  of  the  epidermis.  It  is  now  clear  why  I  have  proposed  to 
call  it  the  Staphylpcoccus  epidermidis  albus.  It  possesses  such  feeble  pyo- 
genic  capacity,  as  is  shown  by  its  behavior  in  wounds  as  well  as  by  experi- 
ments oil  rabbits,  that  the  designation  Staphylococcus  pyogenes  albus  does 
not  seem  appropriate.  Still,  I  am  not  inclined  to  insist  too  much  upon  this 
point,  as  very  probably  this  coccus,  which  has  hitherto  been  unquestionably 
identified  by  Bossowski  and  others  with  the  ordinary  Staphylococcus  pyo- 
genes albus  of  Rosenbach,  is  an  attenuated  or  modified  form  of  the  latter 
organism,  although,  as  already  mentioned,  it  presents  some  points  of  differ- 
ence from  the  classical  description  of  the  white  pyogenic  coccus." 

According  to  Welch,  this  coccus  differs  from  Staphylococcus  pyo- 
genes aureus  not  only  in  color,  but  also  in  the  fact  that  it  liquefies 
gelatin  more  slowly,  does  not  so  quickly  cause  coagulation  of  milk, 
and  is  far  less  virulent  when  injected  into  the  circulation  of  rabbits. 
It  has  been  shown  by  the  researches  of  Bossowski  and  of  Welch 
that  this  coccus  is  very  frequently  present  in  aseptic  wounds,  and 
that  usually  it  does  not  materially  interfere  with  the  healing  of 
wounds,  although  sometimes  it  appears  to  cause  suppuration  along 
the  drainage  tube,  and  it  is  the  usual  cause  of  "  stitch  abscess." 
Bossowski,  in  fifty  cases  of  wounds  treated  antiseptically,  obtained 
bacteria  from  the  discharges  in  forty,  and  in  twenty-six  of  these 
cases  he  found  Staphylococcus  pyogenes  albus  ;  Staphylococcus  au- 
reus was  found  nine  times,  Streptococcus  pyogenes  in  two,  and  vari- 
ous non-pathogenic  bacteria  in  eight.  In  forty-five  laparotomy 
wounds  examined  by  Ghrisky  and  Robb,  in  which  strict  antiseptic 
precautions  had  been  observed,  bacteria  were  found  in  thirty-one,  and 
in  nineteen  of  this  number  Staphylococcus  albus  was  present, 
Staphylococcus  aureus  in  five,  Bacillus  coli  communis  in  six,  and 
Streptococcus  pyogenes  in  three. 

STAPHYLOCOCCUS   PYOGENES   CITREUS. 

Isolated  by  Passet  (1885)  from  the  pus  of  acute  abscesses.  In  thirty - 
three  cases  examined  it  was  found  associated  with  Staphylococcus  albus  in 
two  and  with  Staphylococcus  albus  and  Streptococcus  pyogenes  in  one. 

In  its  morphology  this  coccus  is  identical  with  the  two  preceding  species, 
from  which  it  is  distinguished  by  the  formation  of  a  lemon-yellow  pigment] 
instead  of  a  golden  or  orange- yellow  as  in  Staphylococcus  aureus.  The 
pigment  is  only  formed  in  the  presence  of  oxygen.  This  coccus  is  said  by 
Frankel  to  liquefy  gelatin  more  slowly  than  the  previously  described  species 
— Staphylococcus  aureus  and  Staphylococcus  albus. 

As  to  its  pathogenic  properties  we  have  no  definite  information.  It  is 
included  among  the  pyogenic  bacteria  because  of  its  occasional  presence  in 


382  PYOGENIC   BACTERIA. 

the  pus  of  acute  abscesses,  although  it  has  heretofore  only  been  found  in  as- 
sociation with  other  microorganisms. 

MICROCOCCUS   PYOGENES   TENUIS. 

Obtained  by  Rosenbach  (1884)  from  pus  in  three  cases  out  of  thirty- nine 
examined. 

Morphology. — Micrococci,  somewhat  irregular  in  size,  but  larger  than 
Staphylococcus  albus,  and  seldom  associated  in  masses.  Frequently  the  in- 
dividual cocci  present  the  appearance  of  consisting  of  two  deeply  stained 
masses  separated  from  each  other  by  a  paler  interspace.  Cultures  upon  the 
surface  of  nutrient  agar  form  a  very  thin,  transparent  layer  of  about  one 
millimetre  in  breadth  along  the  line  of  inoculation ;  this  resembles  a  thin 
layer  of  varnish. 

Pathogenesis  undetermined.  (Micrococcus  pneumoiiiae  crouposae  ?) 

STREPTOCOCCUS   PYOGENES. 

Synonyms. — Micrococcus  of  erysipelas  (Fehleisen) ;  Streptococcus 
erysipelatos  ;  Streptococcus  of  pus  ;  Streptococcus  longus  (Yon  Lin- 
gelsheim). 

Obtained  by  Fehleisen  from  the  skin  involved  in  cases  of  erysipe- 
las (1883),  and  by  Rosenbach  (1884)  and  Passet  (1885)  from  the  pus 
of  acute  abscesses.  The  characters  of  the  "  streptococcus  of  erysipe- 
las" of  Fehleisen  and  the  "  Streptococcus  pyogenes"  of  Rosenbach 
and  Passet  are  generally  admitted  to  be  identical,  although  some 
bacteriologists  still  describe  them  separately  and  cultures  from  the 
two  sources  are  still  retained  in  bacteriological  laboratories  under  the 
names  originally  given  them. 

Rosenbach  found  Streptococcus  pyogenes  alone  in  fifteen  cases, 
and  associated  with  staphylococci  in  five  cases,  out  of  thirty-nine 
cases  examined  of  acute  pus  formation.  Passet,  in  thirty-three 
similar  cases,  obtained  the  streptococcus  alone  in  eight  and  associated 
with  staphylococci  in  two.  Subsequent  researches  show  that  this 
micrococcus  is  frequently,  if  not  constantly,  present  in  puerperal 
metritis  ;  that  it  is  the  most  frequent  microorganism  associated  with 
ulcerative  endocarditis  ;  that  it  is  frequently  present  in  diphtheritic 
false  membranes,  and  especially  in  those  cases  of  diphtheritic  inflam- 
mation which  are  secondary  to  scarlet  fever  and  measles  (Prudden). 
Numerous  investigations  made  by  bacteriologists  during  the  past  few 
years  indicate  that  this  is  a  very  important  and  widely  distributed 
pathogenic  microorganism.  It  has  also  been  frequently  found  upon 
exposed  mucous  surfaces — mouth,  nose,  vagina — of  healthy  in- 
dividuals. 

According  to  the  researches  (1891)  of  Von  Lingelsheim,  the  Strep- 
tococcus pyogenes  differs  from  Streptococcus  erysipelatos  in  be- 
ing pathogenic  both  for  mice  and  rabbits,  while  the  latter  is  patho- 


PYOGENIC   BACTERIA.  383 

genie  for  rabbits  only.  The  author  named,  as  a  result  of  extended  and 
carefully  conducted  comparative  studies,  arrives  at  the  following 
conclusions: 

"  According  to  my  observations,  there  are  two  great  groups  among  the 
streptococci.  These  cannot  be  distinguished  one  from  the  other  in  cultures 
in  highly  albuminous  media  (pus,  blood  serum),  but  present  constant  dif- 
ferences when  cultivated  in  bouillon.  The  decisive  characteristics  in  this 
medium  are  :  macroscopic,  the  cloudiness  of  the  medium  ;  microscopic,  the 
length  of  the  chains.  The  two  groups  are  with  difficulty  distinguished  in 
agar  cultures ;  more  easily  in  gelatin,  in  which  the  streptococcus  which 
forms  short  chains  causes  a  slight  liquefaction,  while  the  Streptococcus 
longus  does  not.  Upon  potato  Streptococcus  brevis  alone  shows  a  visible 
growth.  .  .  .  We  see  here  a  group  of  streptococci  which  we  separate  from 
the  others,  because  of  their  microscopic  and  cultural  differences,  under  the 
name  of  Streptococcus  brevis,  which  is  also  distinguished  by  having  no 
pathogenic  action  upon  the  animals  usually  experimented  upon.  We 
recognize,  on  the  other  hand,  the  streptococci  which  we  have  grouped  to- 
gether as  Streptococcus  longus  as  all  pathogenic  and  about  in  equal  degree 
for  a  certain  species  of  animal  (rabbits) ;  but  by  experiments  upon  other 
species  (mice)  we  arrive  at  the  conclusion  that  there  must  also  be  differences 
between  these  streptococci.  It  appears  that  the  streptococci  which  are  dis- 
tinguished by  their  high  degree  of  pathogenic  power  upon  mice  are  also 
those  which  are  distinguished  in  bouillon  cultures  by  the  formation  of  con- 
glomerate masses.  We  find  among  these  also  one  which  is  distinguished 
by  especial  virulence  for  mice,  and  that  this  one  is  distinguished  in  cultures 
by  its  scanty  growth  upon  ox  serum." 

The  more  recent  researches  of  Knorr  (1893),  and  of  Waldvogel 
(1894),  indicate  that  the  classification  of  the  streptococci  proposed  by 
von  Lingelsheim  has  no  great  value,  and  show  that  marked  changes 
in  biological  characters  and  in  pathogenic  power  may  result  from 
cultivation  in  special  media,  or  from  successive  inoculations  into 
animals. 

Morphology. — Spherical  cocci,  from  0.4  //  to  1>  in  diameter,  but 
varying  considerably  in  dimensions  in  different  cultures,  and  even 
in  a  single  chain.     Multiply  by  binary  division, 
in  one  direction  only,  forming  chains,  in  which 
the  elements  are  commonly  associated  in  pairs. 
Under  certain  circumstances,  instead  of  form- 
ing  chains,    a   culture  may  contain    only,    or 

chiefly,  diplococci :  but  usually  chains  contain- 

* 
ing  from  four  to  twenty  or  more  elements  are 

formed,  and    these  are    frequently  associated 

in  tangled  masses.     Occasionally  one  or  more 

cells  in  a  chain  greatly  exceed  their  fellows  in 

size,    and    some    bacteriologists  suppose  that        FIG.  82.— PUS  containing 

these  cells  serve  as  reproductive  spores — arthro-          (jS^00 

spores — but  this  has  not  been  definitely  proven. 

Stains  readily  with  the  aniline  colors  and  by  Gram's  method. 


384 


PYOGENIC   BACTERIA. 


Biological  Characters. — Grows  readily  in  various  liquid  and 
solid  culture  media,  including  all  of  those  usually  employed  in  bac- 
teriological researches.  The  most  favorable  temperature  for  its  de- 
velopment is  from  30°  to  37°  C.,  but  it  multiplies  freely  at  the  ordi- 
nary room  temperature — 16°  to  18°  C. 

Streptococcus  pyogenes  is  a  facultative  anaerobic,  growing 
both  in  the  presence  and  absence  of  oxygen.  It 
does  not  liquefy  gelatin,  and  in  gelatin  stab 
cultures  it  grows  along  the  line  of  puncture, 
forming  numerous  small,  spherical,  translu- 
cent, whitish  colonies,  which  are  closely  crowd- 
ed together  at  the  upper  portion  of  the  line  of 
growth,  and  often  distinctly  separated  from 
each  other  below ;  upon  the  surface  there  is 
often  no  growth,  or  a  scanty  development  may 
occur  about  the  point  of  entrance  of  the  inocu- 
lating needle.  The  minute  colonies  along  the 
line  of  puncture  are  already  visible  at  the  end 
of  twenty -four  hours  in  cultures  kept  in  the 
incubating  oven  at  30°  to  35°  C.,  and  at  the  end 
of  three  or  four  days  they  have  reached  their 
full  development,  forming  a  semi-opaque,  white, 
granular  column,  upon  the  margins  of  which 
the  separate  colonies  are  seen  projecting  into  the 
gelatin.  On  gelatin  plates  very  small,  translu- 
cent colonies  are  developed,  which  upon  the  sur- 
face spread  out  to  form  a  flat,  transparent  disc 
of  about  one-half  millimetre.  Under  a  low  mag- 
nifying power  these  colonies  are  seen  to  be  slight- 
ly granular  and  have  a  yellowish  color.  At  a 
later  date  they  become  darker  and  less  trans- 
parent, and  the  margin  may  show  irregular  projections  made  up  of 
tangled  masses  of  cocci  in  chains.  The  characters  of  growth  in 
nutrient  agar  and  in  jellified  blood  serum  are  similar  to  those  in  gela- 
tin, and  on  agar  plates  colonies  are  formed  similar  to  those  above 
described,  except  that  they  are  somewhat  smaller  and  more  trans- 
parent. Fehleisen  and  De  Simone  state  that  the  erysipelas  coccus' 
may  develop  upon  the  surface  of  cooked  potato,  but  most  authorities 
-Flugge,  C.  Frankel,  Passet,  Baumgarten — agree  that  no  growth 
occurs  upon  potato.  Milk  is  a  favorable  medium  for  the  growth  of 
this  micrococcus,  and  the  casein  is  coagulated  by  it.  A  slightly  acid 
reaction  of  the  culture  medium  does  not  prevent  its  development. 
The  thermal  death-point,  as  determined  by  the  writer,  is  between 
52°  and  54°  C.,  the  time  of  exposure  being  ten  minutes.  According 


FIG.  83.— Streptococcus 
of  erysipelas  iii  nutrient 
gelatin;  stick  culture  at 
end  of  four  days  at  16°- 
18°  C.  (Baumgarten). 


PYOGENIC   BACTERIA. 


385 


to  De  Simone,  a  temperature  of  39.5°  to  41°  C.  maintained  for  two 
days  is  fatal  to  this  micrococcus. 

Manfredi  and  Traversa  have  injected  filtered  cultures  into  frogs, 
guinea-pigs,  and  rabbits  for  the  purpose  of  ascertaining  if  any  solu- 
ble toxic  substance  is  produced  during  the  growth  of  Streptococcus 
pyogenes.  They  report  that  in  some  cases  convulsions  and  in  others 
paralysis  resulted  from  these  injections. 

Von  Lingelsheim  has  (1891)  reported  the  following  results 
obtained  in  an  extended  series  of  experiments  made  to  determine 
the  germicidal  power  of  various  chemical  agents  as  tested  upon 
this  microorganism — time  of  exposure  two  hours  :  Hydrochloric  acid 
1  : 250,  sulphuric  acid  1  : 250,  caustic  soda  1  : 130,  ammonia  1  : 25, 
mercuric  chloride  1  : 2,500,  sulphate  of  copper  1  : 200,  chloride  of 
iron  1  :  500,  terchloride  of  iodine  1  :  750,  peroxide  of  hydrogen  1  :  50, 
carbolic  acid  1  : 300,  cresol  1  : 250,  lysol  1  : 300,  creolin  1  : 130,  naph- 
thylamin  1  : 125,  malachite  green  1  :  3,000,  pyoktanin  1  :  700. 


FIG.  84.— Section  from  margin  of  an  erysipelatous  inflammation,  showing  streptococci  in 
lymph  spaces.    From  a  photograph  by  Koch,    x  900. 

Pathogenesis. — When  inoculated  into  the  cornea  of  rabbits 
Streptococcus  pyogenes  gives  rise  to  keratitis.  Inoculations  into  the 
ear  of  the  same  animal  usually  give  rise  to  a  localized  erysipelatous 
inflammation  accompanied  by  an  elevation  of  temperature  in  the  in- 
oculated ear ;  at  the  end  of  thirty-six  to  forty-eight  hours  the  in- 
flamed area,  which  has  well-defined  margins  and  a  bright-red  color, 
extends  from  the  point  of  inoculation  along  the  course  of  the  veins  to 
the  root  of  the  ear.  This  appearance  passes  away  in  the  course  of  a 
few  days  and  the  animal  recovers.  Subcutaneous  injections  into  mice 
or  rabbits  are  usually  without  result,  and  the  last-named  animal  also 
withstands  injections  of  considerable  quantities  into  the  general  cir- 
culation through  a  vein.  When,  however,  the  animal  has  previously 
been  weakened  by  the  injection  of  toxic  substances  the  streptococcus 
may  multiply  in  its  body  and  cause  its  death  (Fliigge). 

Fehleisen  has  inoculated  cultures,  obtained  in  the  first  instance 
from  the  skin  of  patients  with  erysipelas,  into  patients  in  hospital 
suffering  from  lupus  and  carcinoma,  and  has  obtained  positive  re- 
sults, a  typical  erysipelatous  inflammation  having  developed 
25 


386  PYOGENIC   BACTERIA. 

around  the  point  of  inoculation  after  a  period  of  incubation  of  from 
fifteen  to  sixty  hours.  This  was  attended  with  chilly  sensations  and 
an  elevation  of  temperature.  Persons  who  had  recently  recovered 
from  an  attack  of  erysipelas  proved  to  be  immune. 

Sections  made  from  the  ear  of  an  inoculated  rabbit,  or  of  skin  taken 
from  the  affected  area  in  erysipelas  in  man,  show  the  streptococci  in 
considerable  numbers  in  the  lymph  channels,  but  not  in  the  blood 
vessels.  They  are  more  numerous,  according  to  Koch  and  to  Fehl- 
eisen,  upon  the  margins  of  the  erysipelatous  area,  and  may  even  be 
seen  in  the  lymph  channels  a  little  beyond  the  red  margin  which 
marks  the  line  of  progress  of  the  infection. 

The  researches  of  Weichselbaum  and  others  show  that  Strepto- 
coccus pyogenes  is  the  infecting  microorganism  in  a  certain  propor- 
tion of  the  cases  of  ulcerative  endocarditis.  The  author  named 
found  it  in  four  cases  out  of  fifteen  examined,  and  in  two  cases  of 
endocarditis  verrucosa  out  of  thirteen.  In  a  previously  reported  series 
of  sixteen  cases  (fourteen  of  ulcerative  endocarditis  and  two  of  ver- 
rucosa) the  streptococcus  was  found  in  six. 

In  diphtheritic  false  membranes  this  streptococcus  is  very  com- 
monly present,  and  in  certain  cases  attended  with  a  diphtheritic  exu- 
dation, in  which  the  Bacillus  diphtherias  has  not  been  found  by  com- 
petent bacteriologists,  it  seems  probable  that  Streptococcus  pyogenes 
is  the  pathogenic  microorganism  responsible  for  the  local  inflamma- 
tion and  its  results.  Thus  in  a  series  of  twenty-four  cases  studied  by 
Prudden  in  1889  the  bacillus  of  Loffler  was  not  found,  "but  a  strep- 
tococcus apparently  identical  with  Streptococcus  pyogenes  was  found 
in  twenty-two."  Chantemesse  and  Widal  have  also  reported  cases 
in  which  a  fibrinous  exudate  resembling  that  of  diphtheria  was  as- 
sociated with  a  streptococcus.  "  These  forms  of  so-called  diphtheria 
are  most  commonly  associated  with  scarlatina  and  measles,  erysipe- 
las, and  phlegmonous  inflammation,  or  occur  in  individuals  exposed 
to  these  diseases  ;  but  whether  exclusively  under  these  conditions  is 
not  yet  established"  (Prudden). 

Loffler  has  described  under  the  name  of  Streptococcus  articu- 
lorum  a  micrococcus  obtained  by  him  from  the  affected  mucous 
membrane  in  cases  of  diphtheria,  and  which  he  believes  to  be  acci- 
dentally present  and  without  any  etiological  import  in  this  disease. 
In  its  characters  it  closely  resembles  Streptococcus  pyogenes  and  is 
perhaps  a  variety  of  this  widely  distributed  species.-  Its  characters 
are  described  by  Fliigge  as  follows  : 


"  Cultivated  in  nutrient  gelatin,  it  forms  at  the  end  of  three  days  small, 
transparent,  light-gray  drops,  upon  the  margin  of  which,  under  the  micro- 
scope, the  cocci  in  twisted  chains  may  be  observed.  As  many  as  one  hun- 


PYOGENIC   BACTERIA.  387 

dred  elements  may  be  found  in  a  single  chain,  and  some  of  these  are  distin- 
guished by  their  size ;  occasionally  whole  chains  are  made  up  of  these  large 
cocci,  and  when  closely  observed  some  of  these  may  present  indications  of 
division  transversely  to  the  axis  of  the  chain.  Subcutaneous  inoculation  of 
cultures  into  mice  results  in  the  death  of  a  considerable  number  of  these  ani- 
mals — more  than  half ;  and  the  streptococci  are  found  in  the  spleen  and  other 
organs.  Inoculation  into  the  ear  of  rabbits  causes  an  erysipelatous  inflam- 
mation. When  injected  into  the  circulation  of  these  animals  through  a  vein 
joint  affections  are  developed  in  from  four  to  six  days,  and  a  purulent  ac- 
cumulation occurs  in  which  the  streptococci  are  found.  In  two  rabbits  in- 
oculated in  the  same  way  with  a  culture  of  the  streptococcus  of  erysipelas, 
Loffler  has  observed  a  similar  result." 

Numerous  researches  indicate  that  infection  by  Streptococcus 
pyogenes  through  the  endometrium  is  the  usual  cause  of  puerperal 
fever.  Thus  Clivio  and  Monti  demonstrated  its  presence  in  five 
cases  of  puerperal  peritonitis.  Czerniewski  found  it  in  the  lochia  of 
a  large  number  (thirty-five  out  of  eighty-one)  of  women  suffering 
from  puerperal  fever,  but  in  the  lochia  of  fifty-seven  healthy  puer- 
peral women  he  was  only  able  to  find  it  once.  In  ten  fatal  cases  he 
found  it  in  every  instance,  both  in  the  lochial  discharge  during  life 
and  in  the  organs  after  death.  Widal  carefully  studied  a  series  of 
sixteen  cases  and  arrived  at  the  conclusion  that  this  was  the  infect- 
ing microorganism  in  all.  Bumm  and  other  observers  have  given 
similar  evidence.  Eiselsberg  and  Emmerich  have  succeeded  in  de- 
monstrating the  presence  of  the  streptococcus  in  hospital  wards  con- 
taining cases  of  erysipelas.  That  puerperal  fever  may  result  from 
infection  through  the  finger  of  the  accoucheur,  when  he  has  previ- 
ously been  in  contact  with  cases  of  erysipelas,  has  long  been  taught, 
and,  in  view  of  the  facts  above  recorded,  is  not  difficult  to  under- 
stand. But  in  view  of  the  fact  that  the  streptococcus  of  pus  has  been 
found  in  vaginal  mucus  and  in  the  buccal  and  nasal  secretions  of 
healthy  persons,  it  may  appear  strange  that  cases  of  puerperal  fever 
not  traceable  to  infection  from  erysipelas  or  from  preceding  cases 
do  not  occur  more  frequently.  This  is  probably  largely  due  to  an 
attenuation  of  the  pathogenic  power  of  the  streptococcus  when  it 
leads  a  saprophytic  existence.  Widal  asserts  that,  when  cultivated 
in  artificial  media  for  a  few  weeks,  the  cultures  no  longer  have  their 
original  virulence,  and  Bumm  has  made  the  same  observation.  On 
the  other  hand,  in  "  streptococcus-peritonitis  "  occurring  as  a  result 
of  puerperal  infection  Bumm  states  that  the  thin,  bright-yellow, 
odorless  fluid  contained  in  the  cavity  of  the  abdomen  is  extremely 
virulent ;  a  very  slight  trace,  a  fragment  of  a  drop,  injected  into  the 
abdominal  cavity  of  a  rabbit,  is  sufficient  within  twenty-four  hours 
to  cause  a  general  septic  inflammation  with  a  bloody  serous  exuda- 
tion, quickly  terminating  in  the  death  of  the  animal ;  injected  sub- 
cutaneously  it  gives  rise  to  an  enormous  phlegmon  which  also 


388  PYOGENIC   BACTERIA. 

quickly  proves  fatal.  But  cultures  of  Streptococcus  pyogenes,  after 
it  has  been  carried  through  successive  generations  in  artificial  media, 
injected  beneath  the  skin  of  a  rabbit,  usually  produce  no  result,  or 
at  most  an  abscess  of  moderate  dimensions. 

It  seems  probable  that  the  micrococcus  isolated  by  Fliigge  from 
necrotic  foci  in  the  spleen  of  a  case  of  leucocythsemia,  and  described 
by  him  under  the  name  of  Streptococcus  pyogenes  malignus,  was 
simply  a  very  pathogenic  variety  of  the  streptococcus  of  pus.  He 
was  not  able  to  differentiate  it  from  Streptococcus  pyogenes  by  its 
morphology  or  growth  in  culture  media,  but  it  proved  far  more 
pathogenic  when  tested  upon  animals.  Mice  inoculated  subcutane- 
ously  with  a  minute  quantity  of  a  pure  culture  died,  without  excep- 
tion, in  three  to  five  days.  A  large  abscess  was  formed  at  the  point 
of  inoculation,  and  the  blood  of  the  animal  contained  numerous  cocci 
in  pairs  and  chains.  Rabbits  inoculated  in  the  ear  showed  at  first 
the  same  local  appearances  as  result  from  inoculations  with  strepto- 
coccus of  pus  and  of  erysipelas,  but  after  two  or  three  days  symp  • 
toms  of  general  infection  were  developed,  and  death  occurred  at  the 
end  of  three  or  four  days.  At  the  autopsy  the  cocci  were  found  in 
the  blood,  and  frequently  there  were  purulent  collections  in  the 
joints  containing  the  same  microorganism.  Krause  has  also  de- 
scribed a  streptococcus  which  only  differs  from  Streptococcus  pyo- 
genes of  Rosenbach  and  Passet  by  the  greater  virulence  manifested 
by  its  cultures. 

The  fact  that  pathogenic  bacteria  may  attain  an  intensified  de- 
gree of  virulence  by  cultivation  in  the  bodies  of  susceptible  animals 
was  demonstrated  by  Davaine  many  years  ago,  and  is  fully  estab- 
lished by  the  experiments  of  Pasteur  and  others.  It  is  true  of  the 
anthrax  bacillus,  of  the  writer's  Micrococcus  Pasteuri,  and  of  other 
well-known  pathogenic  microorganisms.  The  reverse  of  this — at- 
tenuation of  virulence  as  a  result  of  cultivation  in  artificial  media — 
is  also  well  established  for  several  pathogenic  species.  Now  it 
appears  that  the  attenuated  streptococcus  is  far  less  likely  to  give 
rise  to  erysipelas  or  to  puerperal  infection  than  is  the  same  micro- 
organism as  obtained  from  a  case  of  one  or  the  other  of  these  infec- 
tious diseases.  The  same  is  probably  true  also  of  Staphylococcus 
aureus  and  other  facultative  parasites  which  are  found  as  sapro- 
phytes upon  the  surface  of  the  body  and  upon  exposed  mucous  mem- 
branes in  healthy  persons.  And  it  is  not  improbable  that  attenuated 
varieties  of  these  micrococci  which  find  their  way  into  open  wounds, 
or  into  the  uterine  cavity  shortly  after  parturition,  if  they  escape 
destruction  by  the  sanguineous  discharge,  acquire  increased  patho- 
genic power  from  their  multiplication  in  it,  as  a  result  of  which  they 
are  able  to  invade  the  living  tissues.  But  it  appears  probable  that 


PYOGENIC   BACTERIA.  389 

• 

infection  through  open  wounds  does  not  depend  alone  upon  the 
potency  of  the  pathogenic  micrococci  present  in  them,  but  also  upon 
the  absorption  of  chemical  poisons  produced  by  septic  (putrefactive) 
bacteria,  which  weaken  the  vital  resisting  power  of  the  tissues. 
Gottstein,  as  a  result  of  experiments  made  by  him,  is  of  the  opinion 
that  the  resorption  of  broken-down  red  blood  corpuscles  favors  infec- 
tion by  pathogenic  bacteria  present  in  wounds  ;  and  he  has  shown 
that  the  injection  into  animals  of  certain  toxic  substances  which  de- 
stroy the  red  corpuscles  in  the  circulation  makes  them  susceptible  to 
the  pathogenic  action  of  certain  bacteria  which  are  harmless  for 
them  under  ordinary  circumstances.  Thus  a  guinea-pig,  an  animal 
which  is  immune  against  the  bacillus  of  fowl  cholera,  succumbed  to  an 
inoculation  made  after  first  injecting  subcutaneously  0.06  gramme  of 
hydracetin  dissolved  in  alcohol.  At  the  autopsy  hsemorrhagic  exu- 
dations were  found  in  the  serous  cavities,  hsemorrhagic  infarctions 
in  the  lungs,  and  quantities  of  the  bacillus  injected  were  found  in 
the  blood  and  in  fluid  from  the  cavity  of  the  abdomen. 

In  man  the  ever-present  pus  cocci  are  more  likely  to  invade  the 
tissues,  forming  furuncles,  carbuncles,  and  pustular  skin  eruptions, 
or  erysipelatous  and  phlegmonous  inflammations,  when  the  standard 
of  health  is  reduced  from  any  cause,  and  especially  when  by  absorp- 
tion or  retention  various  toxic  organic  products  are  present  in  the 
body  in  excess.  It  is  thus  that  we  would  explain  the  liability  to  these 
local  infections,  as  complications  or  sequelae  of  various  specific  infec- 
tious diseases,  in  the  victims  of  chronic  alcoholism,  in  those  exposed 
to  septic  emanations  from  sewers,  etc. ,  and  probably  in  many  cases 
from  the  absorption  of  toxic  products  formed  in  the  alimentary  canal 
as  a  result  of  the  ingestion  of  improper  food,  or  of  abnormal  fermen- 
tative changes  in  the  contents  of  the  intestine,  or  from  constipation. 

The  Pus  Cocci  in  Inflammations  of  Mucous  Membranes. — 
To  what  extent  the  pus  cocci  are  responsible  for  inducing  and  main- 
taining non-specific  inflammations  of  mucous  membranes  has  not 
been  determined  ;  but  having  demonstrated  the  pyogenic  properties 
of  these  cocci,  their  presence  in  the  purulent  discharges  from  inflamed 
mucous  membranes  can  scarcely  be  considered  as  unimportant,  not- 
withstanding the  fact  that  they  are  also  frequently  found  in  secre- 
tions from  healthy  mucous  surfaces.  They  are  likewise  found  upon 
the  skin  of  healthy  persons,  and  yet  we  have  unimpeachable  experi- 
mental evidence  that  they  may  produce  a  local  inflammation,  at- 
tended with  pus  formation,  when  injected  subcutaneously,  or  even 
when  freely  applied  to  the  uninjured  surface. 

In  otitis  media  Levy  and  Schrader  obtained  Staphylococcus 
albus  in  pure  cultures  in  three  cases  out  of  ten  in  which  paracentesis 
was  performed,  and  in  two  others  it  was  present  in  association  with 


390  PYOGENIC   BACTERIA. 

• 

other  microorganisms.  In  eighteen  cases  of  otitis  media  in  young 
children  Netter  found  Staphylococcus  aureus  six  times  and  Strepto- 
coccus pyogenes  thirteen  times.  Scheibe,  in  eleven  cases  in  which 
perforation  had  not  yet  taken  place,  found  Staphylococcus  albus  in 
two  and  various  other  microorganisms  in  the  remaining  cases  ;  Sta- 
phylococcus aureus  was  not  present  in  any.  Habermann  obtained 
aureus  associated  with  other  bacteria  in  a  single  case  of  purulent 
otitis  media.  In  a  series  of  eight  cases  occurring  as  a  sequela  of 
influenza  Scheibe  obtained  Streptococcus  pyogenes  in  two,  "  diplo- 
coccus  pneumonias"  in  two,  Staphylococcus  aureus  in  one,  Strepto- 
coccus pyogenes  and  Staphylococcus  albus  together  in  two,  and  Strep- 
tococcus pyogenes  in  association  with  an  undescribed  micrococcus  in 
one.  In  all  of  these  cases  a  slender  bacillus  was  also  present,  as 
shown  by  microscopical  examination,  which  did  not  grow  in  any  of 
the  culture  media  employed.  Bordoni-Uffreduzzi  and  Gradenigo 
have  tabulated  the  results  obtained  by  various  bacteriologists  who 
have  examined  pus  obtained  through  the  previously  intact  tympanic 
membrane.  In  thirty-two  cases  of  this  character  the  microorganism 
most  frequently  found  was  diplococcus  pneumoniae  (Micrococcus 
pneumonise  crouposse  of  the  present  writer),  which  was  present  in  a 
pure  culture  in  thirteen  and  associated  with  Staphylococcus  aureus 
in  one,  with  Staphylococcus  albus  in  one,  and  with  Streptococcus 
pyogenes  in  one.  In  the  other  sixteen  cases  the  pyogenic  cocci  were 
present  in  all  but  two,  in  which  bacilli  were  found — Bacillus  tenuis 
in  one,  a  non-liquefying  bacillus  in  one.  In  twenty-seven  cases  in 
which  the  pus  was  withdrawn  from  one  to  thirty  days  after  paracen- 
tesis  or  spontaneous  rupture  of  the  membrane,  the  pyogenic  cocci 
were  present  in  twenty  and  diplococcus  pneumoniae  in  seven. 

In  acute  nasal  catarrh  Paulsen  found  Staphylococcus  aureus  in 
seven  cases  out  of  twenty-four  examined,  and  E.  Frankel  in  two  out  of 
four  ;  but  it  must  be  remembered  that  Von  Besser  has  shown  that  this 
micrococcus  is  frequently  present  in  the  secretions  from  the  healthy 
nasal  mucous  membrane,  and  we  have  experimental  evidence  that 
the  pus  organisms,  when  introduced  into  the  conjunctival  sac  of 
rabbits  (Widmark),  do  not  give  rise  to  catarrhal  inflammation.  On 
the  other  hand,  Widmark  found  that  when  inoculated  into  the  cornea 
of  rabbits  an  intense  conjunctivitis  resulted,  together  with  keratitis 
and  perforation  of  the  cornea  in  fifteen  per  cent  of  the  cases.  The 
same  author  in  his  bacteriological  researches  obtained  the  pyogenic 
staphylococci  from  the  circumscribed  abscesses  of  blepharadenitis, 
while  in  inflammation  of  the  lacrymal  sac  Streptococcus  pyogenes 
was  usually  present. 

Shougolowicz,in  the  bacteriological  examination  of  twenty-six  cases 
of  trachoma,  found  Staphylococcus  albus  in  twelve,  Staphylococcus 


PYOGENIC   BACTERIA.  391 

aureus  in  nine,  Staphylococcus  citreus  in  three,  and  Staphylococcus 
cereus  albus  in  three.  These  pus  organisms  were  in  a  number  of 
the  cases  associated  with  other  well-known  saprophytes,  and  in  seven 
cases  a  short  bacillus  not  previously  described  was  found.  That 
various  bacilli  are  found  in  the  conjunctival  sac  of  healthy  eyes 
and  in  different  forms  of  conjunctivitis  has  been  shown  by  Fick, 
whose  results  do  not  correspond  in  this  respect  with  those  of  Gif- 
ford,  who  found  almost  exclusively  micrococci.  Whatever  may  be 
the  final  conclusion  as  to  the  role  of  the  pus  cocci  heretofore  de- 
scribed in  the  etiology  of  acute  or  chronic  conjunctivitis,  there  can  be 
no  doubt  of  the  power  of  the  "  gonococcus"  to  induce  a  virulent  in- 
flammation of  the  conjunctive  when  introduced  into  healthy  eyes. 

MICROCOCCUS  GONORRHCE^E. 

Synonym.  —  Gonococcus  (Neisser). 

Discovered  by  Neisser  (1870)  in  gonorrhoea!  pus  and  described  by 
him  under  the  name  of  "  Gonococcus/'    Cultivated  by  Bumm  (1885), 
and  infective  virulence  proved  by  inocula- 
tion into  man.    Constantly  present  in  viru- 
lent gonorrhoea!  discharges,  for  the  most 
part  in  the  interior  of  the  pus  cells  or  at- 
tached to  the  surface  of  epithelial  cells. 

Morphology.  —  Micrococci,  usually  join-      ft 
ed  in  pairs  or  in  groups  of  four,  in  which 
the    elements    are    flattened  -  -  "  biscuit- 
shaped."    The  flattened  surf  aces  face  each 

other  and  are  separated,  in  stained  pre-          ?§   &)    ^    %$    €1 
parations,    by    an    unstained    interspace.  c 

*  .          A  Fia.    85.  —  a,   gonococci  from    a 

Ihe  diameter  of  an  associated  pair  ot  cells  pure  culture,  x  about  1,000  ;  &,gono- 
varies  from  0.8  to  1.6  /i  in  the  long  dia-  cocci  in  PUS  cells  and  epithelial  CPU 

,      ,,  from  case  of  gonorrhoeal   ophthal- 

meter—  average  about  1.2o  /*—  and   from  mia;  c,  form  and  mode  of  division 


0.6   to  0.8  yw  in  the  line  of  the  interspace  of  gonococci-schematic. 
between  the  biscuit-shaped  elements,  which 

sometimes  present  a  slight  concavity  of  the  flattened  surfaces.  Mul- 
tiplication occurs  alternately  in  two  planes,  and  as  a  result  of  this 
groups  of  four  are  frequently  observed.  But  diplococci  are  more 
numerous  and  are  considered  as  the  characteristic  mode  of  grouping. 
Single,  spherical,  undivided  cells  are  rarely  seen. 

It  must  be  remembered  that  the  morphology  of  this  micrococcus 
as  above  described  does  not  suffice  to  distinguish  it,  for  Bumm  has 
shown  that  "  the  biscuit  form  is  not  at  all  specific  for  the  gonococcus, 
but  is  shared  with  it  by  a  number  of  microorganisms,  which  consist 
of  two  hemispherical  elements  with  the  flattened  surfaces  facing  each 


392 


PYOGENIC   BACTERIA. 


other  and  separated  by  a  cleft,  and  some  of  these  correspond  in  their 
morphology,  in  every  detail,  with  the  gonococcus." 

Stains  quickly  with  the  basic  aniline  colors,  especially  with 
methyl  violet,  gentian  violet,  and  fuchsin ;  not  so  quickly  with 
methylene  blue,  which  is,  however,  one  of  the  most  satisfactory 
staining  agents  for  demonstrating  its  presence  in  pus.  Beautiful 
double-stained  preparations  may  be  made  from  gonorrhoeal  pus, 
spread  upon  a  cover  glass  and  "  fixed/'  secundum  art  em,  by  the  use 
of  methylene  blue  and  eosin.  Does  not  stain  by  Gram's  method — 
i.e.,  the  cocci  are  decolorized,  after  having  been  stained  with  an  ani- 
line color,  by  being  immersed  in  the  iodine  solution  employed  in 
Gram's  method  of  staining.  But  this  character  cannot  be  depended 
upon  alone  for  establishing  the  diagnosis,  for  Bumm  has  shown  that 


Fia.  86.—"  Gonococcus  "  in  gonorrhoeal  pus.    From  a  photomicrograph  by  Frankel  and  Pfeiffer. 
X  1,000. 

other  diplococci  are  occasionally  found  in  gonorrhoeal  pus  which  do 
not  stain  by  this  method.  It  serves  to  distinguish  them,  however,  from 
the  common  pus  cocci  heretofore  described — Staphylococcus  aureus, 
Staphylococcus  albus,  Staphylococcus  citreus — which  retain  their 
color  when  treated  in  the  same  way.  A  more  trustworthy  diagnostic 
character  is  that  these  biscuit-shaped  diplococci  are  found  within  the 
pus  cells,  sometimes  one  or  two  pairs  only,  but  more  frequently  in 
considerable  numbers,  and  occasionally  in  such  numbers  as  to  com- 
pletely fill  the  cell.  No  similar  picture  is  presented  by  pus  from  any 
other  source,  with  the  exception  of  that  from  a  form  of  "  puerperal 
cystitis "  which  Bumm  has  described.  But  in  this  the  diplococci 
contained  in  the  pus  cells  were  to  be  distinguished  by  the  fact  that 
they  retained  their  color  when  treated  by  Gram's  method.  Owing 


PYOGENIC   BACTERIA.  393 

to  the  difficulty  of  cultivating  this  micrococcus,  and  the  importance, 
under  certain  circumstances,  of  not  making  a  mistake  in  its  diag- 
nosis, these  characters  are  of  exceptional  value. 

Biological  Characters. — Bumm  (1885)  first  succeeded  in  culti- 
vating the  "  gonococcus "  upon  human  blood  serum,  obtained  from 
the  placenta  of  a  recently  delivered  woman.  He  found  that  the  cul- 
tures thrive  best  in  a  moist  atmosphere  at  30°  to  34°  C.  The  growth 
under  the  most  favorable  conditions  is  slow,  and  frequently  no  devel- 
opment occurs  when  pus  containing  numerous  gonococci  is  placed 
upon  blood  serum  in  an  incubating  oven ;  or  after  a  slight  multi- 
plication development  ceases  and  the  cocci  undergo  degenerative 
changes  and  quickly  disappear. 

Cultures  upon  the  surface  of  blood  serum  form  a  very  thin,  often 
scarcely  visible  layer,  with  a  smooth,  moist,  shining  surface,  and 
by  reflected  light  a  grayish-yellow  color.  The  growth  at  the  end  of 
twenty-four  hours  may  extend  for  a  distance  of  a  millimetre  along 
the  line  of  inoculation,  but  at  the  end  of  two  or  three  days  no  fur- 
ther development  occurs  and  the  cocci  soon  lose  their  vitality.  This 
micrococcus,  then,  is  aerobic.  Whether  it  may  also  be  a  facultative 
anaerobic  has  not  been  definitely  determined,  but  it  does  not  grow 
along  the  line  of  puncture  when  stick  cultures  are  made  in  blood  se- 
rum. Its  rapid  and  abundant  multiplication  in  gonorrhoeal  infection 
of  mucous  membranes,  and  the  difficulties  attending  its  cultivation 
in  artificial  media,  show  that  the  gonococcus  is  a  strict  parasite. 

Lestikow  and  Loffler,  prior  to  the  publication  of  Bumm's  impor- 
tant monograph,  had  reported  successful  results  in  cultivating  the 
gonococcus  upon  a  mixture  of  blood  serum  and  gelatin.  Bockhart 
has  since  recommended  a  mixture  of  nutrient  agar  (two  parts),  lique- 
fied at  a  temperature  of  50°  C.,  with  blood  serum  (two  to  three  parts) 
at  20°  C.  By  quickly  mixing  with  this  a  little  pus  containing  the 
gonococcus  he  was  able  to  obtain  colonies  upon  plate  cultures,  made 
by  pouring  the  liquid  medium  upon  sterile  glass  plates  in  the  usual 
manner. 

Ghon  and  Schlagenhaufer  in  1893  reported  that  they  obtained 
good  results  by  adding  phosphate  of  soda  to  blood-serum  agar,  made 
according  to  the  method  of  Wertheim — one  part  of  human  blood 
serum  from  the  placenta  to  two  or  three  parts  of  nutrient  agar.  Also 
that  they  were  successful  in  cultivating  the  gonococcus  in  an  acid 
medium  made  by  adding  one  part  of  urine  to  two  of  nutrient  agar 
(two  per  cent).  Turro  (1894)  has  since  published  the  results  of  his 
experiments  relating  to  the  cultivation  of  this  micrococcus  in  acid 
media.  According  to  him  it  grows  in  normal  urine,  either  with  or 
without  the  addition  of  peptone  (one  per  cent) ;  also  in  acid  gelatin, 
prepared  in  the  usual  way  but  without  neutralization  (?). 


394 


PYOGENIC   BACTERIA. 


Turro  also  claims  to  have  produced  specific  urethritis  in  dogs  by 
inoculation  with  his  cultures.  Heiman  (1895)  as  a  result  of  an  ex- 
tended experimental  research,  arrives  at  the  conclusion  that  "the 
diplococcus  described  by  Turro  in  connection  with  his  acid  media  is 
not  the  gonococcus."  His  inoculation  experiments  in  dogs,  made 
with  pure  cultures  of  the  gonococcus,  gave  an  entirely  negative  result. 
For  the  cultivation  of  the  gonococcus,  Heiman  recommends  a  medium 
made  from  "chest  serum"  obtained  from  a  patient  suffering  with 
hydrothorax  or  acute  pleurisy.  This  was  found  to  be  superior  to 
placenta  serum,  sheep-blood  serum,  or  peritoneum  serum,  because  of 
the  great  amount  of  serum  albumin  which  it  contains.  Two  per 
cent  of  agar,  one  per  cent  of  peptone,  and  one-half  per  cent  of  sodium 
chloride  were  added  to  the  chest  serum,  and  the  medium  was  sterilized 
by  "fractional  sterilization." 


FIG.  87.— Gonorrhoeal  conjunctivitis,  second  day  of  sickness;  section  through  the  mucous  mem- 
brane of  upper  eyelid;  invasion  of  the  epithelial  layer  by  gonococci.    (Bumm.) 

Schrotter  and  Winkler  (1890)  report  their  success  in  cultivating 
the  gonococcus  upon  albumin  from  the  egg  of  the  pewit — "  Kibitz" 
In  the  culture  oven  at  38°  C.  a  thin,  transparent,  whitish  layer  was 
already  visible  at  the  end  of  six  hours  and  rapidly  extended  ;  the 
growth  was  less  abundant  at  the  end  of  three  days,  and  had  entirely 
ceased  by  the  fifth  day.  Attempts  to  cultivate  the  same  microor- 
ganism in  albumin  from  hens'  eggs  gave  a  negative  result. 

Aufuso  (1891)  has  cultivated  the  gonococcus  in  fluid  obtained 
from  the  knee  joint  in  a  case  of  chronic  synovitis,  but  failed  to  culti- 
vate it  in  the  fluid  of  ascites.  A  culture  of  the  twelfth  generation 
made  upon  the  culture  medium  mentioned,  solidified  by  heat,  was 
introduced  into  the  urethra  of  a  healthy  man  and  gave  rise  to  a 
characteristic  attack  of  gonorrhoea. 

Development  does  not  occur  below  25°  or  above  38°  C.  The 
writer  has  shown  that  a  temperature  of  60°  C.  maintained  for  ten 
minutes  destroys  the  infective  virulence  of  gonorrhoeal  pus. 

Pathogenesis. — That  the  gonococcus  is  the  cause  of  the  specific 
inflammation  and  purulent  discharge  characteristic  of  gonorrhoea  is 
now  generally  admitted  upon  the  experimental  evidence  obtained  by 


PYOGENIC   BACTERIA.  395 

Bumm.  Having  succeeded  in  obtaining  it  in  pure  cultures  from 
gonorrhoeal  pus,  he  made  successful  inoculations  in  the  healthy  ure- 
thra in  two  cases — once  with  a  third  culture  and  once  with  one 
which  had  been  transferred  through  twenty  successive  generations. 
In  both  cases  a  typical  gonorrhoea  developed  as  a  result  of  the  inocu- 
lation. 

The  mucous  membranes  in  man  which  are  subject  to  gonorrhceal 
infection  are  those  of  the  urethra,  the  conjunctiva,  the  cervix  uteri, 
and  the  vagina  in  children — the  vagina  in  adults  is  not  involved. 
Inoculations  of  gonorrhceal  pus  into  the  vagina  or  conjunctival  sac  of 
the  lower  animals — dogs,  rabbits,  horses,  apes — are  without  result. 

The  very  numerous  researches  which  have  been  made  by  compe- 
tent bacteriologists  show  that  the  gonococcus  is  constantly  present  in 
gonorrhoeal  discharges,  and  in  view  of  the  facts  above  stated  its  etio- 
logical  import  appears  to  be  fully  established.  Bumm  has  studied 
the  development  of  blennorrhoea  neonatorum,  and  has  shown  that 
soon  after  infection  the  presence  of  gonococci  may  be  demonstrated 
in  the  superficial  epithelial  cells  of  the  mucous  membrane  and  be- 
tween them  ;  that  they  soon  penetrate  to  the  deeper  layers,  and  that 
by  the  end  of  forty-eight  hours  the  entire  epithelial  layer  is  invaded 
by  the  diplococci,  which  penetrate  by  way  of  the  connecting  mate- 
rial— "  Kittsubstance  "  —between  the  cells.  They  also  multiply  in 
the  superficial  layers  of  connective  tissue  and  give  rise  to  an  inflam- 
matory reaction,  which  is  shown  by  an  abundant  escape  of  leuco- 
cytes from  the  dilated  capillary  network.  The  penetration  of  the 
gonococci  to  the  deeper  layers  of  the  mucous  membrane  of  the  ure- 
thra, and  even  to  the  corpus  cavernosum,  was  observed  by  Bockhart 
in  a  case  studied  by  him  in  which  death  occurred  during  an  acute 
attack  of  gonorrhoea.  But  Bumm  concludes  from  his  researches 
that  this  is  not  usual,  and  that  the  invasion  is  commonly  limited  to 
the  superficial  layers  of  the  mucous  membrane. 

Staphylococcus  pyogenes  aureus  is  not  infrequently  associated 
with  the  gonococcus  in  late  gonorrhoeal  discharges,  and  the  abscesses 
which  occasionally  develop  as  a  complication  of  gonorrhoea,  in  the 
prostate,  the  inguinal  glands,  or  around  the  urethra,  are  probably 
due  to  its  presence,  which  has  been  demonstrated  in  the  pus  from 
such  abscesses  in  a  number  of  cases.  The  same  is  true  of  the  joint 
affections  and  endocarditis  which  sometimes  occur  in  the  course  of 
an  attack  of  gonorrhoea.  Although  some  authors  have  claimed  to 
find  the  gonococcus  in  these  so-called  metastatic  gonorrhceal  inflam- 
mations, the  evidence  is  not  satisfactory,  and  it  seems  probable  that 
the  Staphylococcus  aureus  is  the  usual  microorganism  concerned  in 
these  affections. 


VI. 
BACTERIA  IN  CROUPOUS  PNEUMONIA. 

BACILLUS   OF  FRIEDLANDER. 

Synonyms. — Pneumococcus  (Friedlander);  Bacillus  pneumonia 
(Fliigge). 

Obtained  by  Friedlander  and  Frobenius  in  pure  cultures  (1883) 
from  the  exudate  into  the  pulmonary  alveoli  in  cases  of  croup- 
ous  pneumonia.  Subsequent  researches  show  that  it  is  only  present 
in  a  small  proportion  of  the  cases — nine  times  in  one  hundred  and 
twenty-nine  cases  examined  by  Weichselbaum,  three  times  in  seventy 
cases  examined  by  Wolf. 

Morphology. — Short  rods  with  rounded  ends,  often  so  short  as 

to  resemble  micrococci,  especially  in  very 
recent  cultures  ;  commonly  united  in  pairs 
or  chains  of  four,  and  under  certain  cir- 
cumstances surrounded  by  a  transparent 
capsule.      The    gelatinous  envelope  —  so- 
FIG.  88.— Bacillus  of  Friedlander;     called  capsule — is  not  seen  in  preparations 
a,  from  a  culture;  6,  from  blood  of    ma(je  f rom  cuitures  in  artificial  media,  but 

mouse,  showing  capsule.  (Flugge.)      .  .  . 

is  very  prominent  in  properly  stained  prepa- 
rations from  the  blood  of  an  inoculated  animal.  It  often  has  a  diame- 
ter equal  to  or  greater  than  that  of  the  enclosed  cell,  and  appears  to 
consist  of  a  substance  resembling  mucin,  which  is  soluble  in  water  or 
dilute  alcohol.  Where  several  cells  are  united  in  a  chain  they  may 
all  be  enclosed  in  a  common  envelope,  or  each  may  have  its  own  cap- 
sule. This  capsule  is  not  peculiar  to  Friedlander's  bacillus,  as  he 
at  first  supposed,  but  is  found  in  other  bacilli  and  also  in  the  writer's 
Micrococcus  Pasteuri. 

Friedlander's  bacillus  stains  readily  with  the  aniline  colors,  but 
is  decolorized  by  the  iodine  solution  used  in  Gram's  method.  In 
preparations  from  the  blood  of  an  inoculated  animal,  stained  by  an 
aniline  color,  the  capsule  appears  as  an  unstained  envelope  surround- 
ing the  stained  cell,  but  by  special  treatment  the  capsule  may  also  be 
stained.  Friedlander's  method  is  as  follows  :  The  section  or  cover- 


BACTERIA   IN  CROUPOUS   PNEUMONIA. 


397 


glass  preparation  is  placed  for  twenty-four  hours  in  a  solution  of 
gentian  violet  and  acetic  acid,  containing  fifty  parts  of  a  concentrated 
alcoholic  solution  of  gentian  violet,  one  hundred  parts  of  distilled 
water,  and  ten  parts  of  acetic  acid.  The  stained  preparation  is 
washed  for  a  minute  or  two  in  a  one-per-cent  solution  of  acetic  acid, 
dehydrated  with  alcohol,  cleared  up  with  oil  of  cloves  or  cedar,  and 
mounted  in  balsam.  The  bacillus  is  quickly  stained  in  dried  cover- 
glass  preparations  by  immersion  in  aniline- water-gentian-violet  solu- 
tion (two  or  three  minutes).  The  stained  preparation  should  be  de- 
colorized by  placing  it  in  absolute  alcohol  for  half  a  minute,  and  then 
washed  in  distilled  water. 

Biological  Characters. — This  bacillus  does  not,  so  far  as  is 
known,  form  reproductive  spores ;  it  is  non-motile  and  does  not 
liquefy  gelatin.  It  is  aerobic  and  a  facultative  anaerobic.  In 
gelatin  stab  cultures  it  presents  the  "nail-shaped"  growth  first 
described  by  Friedlander,  which  is  not,  however,  peculiar  to  this 
bacillus.  The  head  of  the  nail  is  formed  by  the 
development  around  the  point  of  entrance  of  the 
inoculating  needle  of  a  rounded,  white  mass  hav- 
ing a  smooth,  shining  surface,  and  its  stem  by  the 
growth  along  the  line  of  puncture.  This  consists 
of  closely  crowded,  opaque,  white,  spherical  colo- 
nies. Gas  bubbles  sometimes  develop  in  gelatin 
cultures,  and  in  old  cultures  the  gelatin  about  the 
line  of  growth  acquires  a  yellowish-brown  color. 
The  growth  in  nutrient  agar  resembles  that  in 
gelatin.  Upon  the  surface  of  blood  serum  abun- 
dant grayish- white,  viscid  masses  are  developed. 
Upon  potato  the  growth  is  abundant,  quickly  cov- 
ering the  entire  surface  with  a  thick,  yellowish- 
white,  glistening  layer  which  often  contains  gas 
bubbles  when  the  temperature  is  favorable.  Col- 
onies in  gelatin  plates  appear  at  the  end  of  twenty- 
four  hours  as  small,  white  spheres,  which  increase 
rapidly  in  size,  and  upon  the  surface  form  round- 
ed, smooth,  glistening,  white  masses  of  consider- 
able size.  Under  the  microscope  the  colonies  pre- 
sent a  somewhat  irregular  outline  and  a  slightly  Fia*  89  ~Friedl5nder1s 

0  y     bacillus;   stab  culture  in 

granular  appearance.  Growth  occurs  at  compara-  gelatin;  end  of  four  days 
tively  low  temperatures— 16°  to  20°  C.— but  is  more  at  16°-18°  °-  (Baumgar- 
rapid  in  the  incubating  oven.  The  thermal  death- 
point,  as  determined  by  the  writer,  is  about  56°  C.  In  the  ordinary 
culture  media  it  retains  its  vitality  for  a  long  time,  and  may  grow 
when  transplanted  to  fresh  culture  material  after  having  been  pre- 


398  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

served  for  a  year  or  more.  At  a  temperature  of  40°  C.  development 
ceases. 

Pathogenesis. — In  Friedlander's  experiments  the  bacillus  from 
pure  cultures,  suspended  in  water,  was  injected  through  the  thoracic 
wall  rinto  the  right  lung  of  dogs,  rabbits,  guinea-pigs,  and  mice. 
Rabbits  proved  to  be  immune ;  one  dog  out  of  five,  six  guinea-pigs 
out  of  eleven,  and  all  of  the  mice  (thirty-two)  succumbed  to  the 
inoculation.  At  the  autopsy  the  pleural  cavities  were  found  to  con- 
tain a  sero-purulent  fluid  ;  the  lungs  were  intensely  congested,  con- 
tained but  little  air,  and  in  places  showed  limited  areas  of  red  infil- 
tration ;  the  spleen  was  considerably  enlarged ;  the  bacillus  was 
found  in  great  numbers  in  the  lungs,  the  fluid  in  the  pleural  cavi- 
ties, and  in  the  blood  obtained  from  the  general  circulation  or  from 
the  various  organs  of  the  body.  Similar  appearances  presented  them- 
selves in  the  case  of  the  guinea-pigs  which  succumbed  to  the  inocu- 
lation. 

These  results  show  that  the  bacillus  under  consideration  is  path- 
ogenic for  mice  and  for  guinea-pigs,  but  they  are  by  no  means 
sufficient  to  prove  that  it  is  capable  of  producing  a  genuine  croupous 
pneumonia  in  man,  and  it  is  still  uncertain  whether  its  occasional 
presence  in  the  exudate  into  the  pulmonary  alveoli  in  cases  of  this 
disease  has  any  etiological  importance. 

MICROCOCCUS   PNEUMONIA   CROUPOS^Q. 

Synonyms. — Micrococcus  Pasteuri  (Sternberg) ;  Micrococcus  of 
sputum  septicaemia  (Frankel)  ;  Diplococcus  pneumonise  (Weichsel- 
baum) ;  Bacillus  septicus  sputigenus  (Fliigge)  ;  Bacillus  salivarius 
septicus  (Biondi)  ;  Lancet-shaped  micrococcus  (Talamon) ;  Strepto- 
coccus lanceolatus  Pasteuri  (Gameleia). 

Discovered  by  the  present  writer  in  the  blood  of  rabbits  inocu- 
lated subcutaneously  with  his  own  saliva  in  September,  1880  ;  by 
Pasteur  in  the  blood  of  rabbits  inoculated  with  the  saliva  of  a  child 
which  died  of  hydrophobia  in  one  of  the  hospitals  of  Paris  in  De- 
cember, 1880  ;  identified  with  the  micrococcus  in  the  rusty  sputum  of 
pneumonia,  by  comparative  inoculation  and  culture  experiments,  by 
the  writer  in  1885  (paper  published  in  the  American  Journal  of  the 
Medical  Sciences,  July  1st,  1885).  Proved  to  be  the  cause  of  croup- 
ous pneumonia  in  man  by  the  researches  of  Talamon,  Salvioli,  Stern- 
berg,  Frankel,  Weichselbaum,  Netter,  Gameleia,  and  others. 

The  Presence  of  Micrococcus  Pasteuri  in  the  Salivary  Secre- 
tions of  Healthy  Individuals. — In  September,  1880,  while  engaged 
in  investigations  relating  to  the  etiology  of  the  malarial  fevers,  I  in- 
jected a  little  of  my  own  saliva  beneath  the  skin  of  two  rabbits  as  a 
control  experiment.  To  my  surprise  the  animals  died,  and  I  fomnd 


BACTERIA   IN   CROUPOUS   PNEUMONIA. 

in  their  blood  a  multitude  of  oval  microorganisms,  united  for  the 
most  part  in  pairs,  or  in  chains  of  three  or  four  elements.  These 
experiments  are  recorded  in  my  paper  entitled  "  Experimental  Inves- 
tigations Relating  to  the  Etiology  of  the  Malarial  Fevers/'  published 
in  the  Report  of  the  National  Board  of  Health  for  1881,  pp.  74,  75. 

Following  up  my  experiments  made  in  New  Orleans  (in  Septem- 
ber, 1880),  in  Philadelphia  (January,  1881),  and  in  Baltimore  (March, 
1881),  I  obtained  the  following  results  : 

"  The  saliva  of  four  students,  residents  of  Baltimore  (in  March), 
gave  negative  results  ;  eleven  rabbits  injected  with  the  saliva  of  six 
individuals  in  Philadelphia  (in  January)  gave  eight  deaths  and  three 
negative  results;  but  in  the  fatal  cases  a  less  degree  of  virulence  was 
shown  in  six  by  a  more  prolonged  period  between  the  date  of  injec- 
tion and  the  date  of  death.  This  was  three  days  in  one,  four  days 
in  four,  and  seven  days  in  one. " 

In  a  paper  published  in  the  Journal  of  the  Royal  Microscopical 
Society  (June,  1886)  I  say  : 

"  My  own  earlier  experiments  showed  that  there  is  a  difference  in 
the  pathogenic  potency  of  the  saliva  of  different  individuals,  and  I 
have  since  learned  that  the  saliva  of  the  same  individual  may  differ 
in  this  respect  at  different  times.  Thus  during  the  past  three  years 
injections  of  my  own  saliva  have  not  infrequently  failed  to  cause  a 
fatal  result,  and  in  fatal  cases  death  is  apt  to  occur  after  a  some- 
what longer  interval,  seventy-two  hours  or  more  ;  whereas  in  my 
earlier  experiments  the  animals  infallibly  died  within  forty-eight 
hours." 

The  presence  of  my  Micrococcus  Pasteuri  was  demonstrated  in 
the  blood  of  the  rabbits  which  succumbed  to  the  inoculations. 

Claxton,  in  a  series  of  experiments  made  in  Philadelphia  in  1882, 
injected  the  saliva  of  seven  individuals  into  eighteen  rabbits.  Five 
of  these  died  within  five  days,  and  nine  at  a  later  period. 

Frankel,  whose  first  publication  was  made  in  1885,  discovered 
the  presence  of  this  micrococcus  in  his  own  salivary  secretions  in  1883, 
and  has  since  made  extended  and  important  researches  with  refe- 
rence to  it.  The  saliva  of  five  healthy  individuals  and  the  sputa 
of  patients  suffering  from  other  diseases  than  pneumonia,  injected 
into  eighteen  rabbits,  induced  fatal  "  sputum  septicaemia  "  in  three 
only.  When  he  commenced  his  experiments  his  saliva  was  uni- 
formly fatal  to  rabbits,  but  a  year  later  it  was  without  effect. 

Wolf  injected  the  saliva  of  twelve  healthy  individuals,  and  of 
three  patients  with  catarrhal  bronchitis,  into  rabbits,  and  induced 
"  sputum  septicaemia  "  in  three. 

Netter  examined  the  saliva  of  one  hundred  and  sixty-five  healthy 
persons,  by  inoculation  experiments  in  rabbits,  and  demonstrated 
the  presence  of  this  micrococcus  in  fifteen  per  cent  of  the  number. 


400  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

Vignal,  in  his  recent  elaborate  paper  upon  the  microorganisms 
of  the  mouth,  says  : 

"  Last  year  I  encountered  this  microbe  continually  in  my  mouth 
during  a  period  of  two  months,  then  it  disappeared,  and  I  did  not 
find  it  again  until  April  of  this  year,  and  then  only  for  fifteen  days, 
when  it  again  disappeared  without  appreciable  cause. " 

The  Presence  of  Micrococcus  Pneumonice  Crouposce  in  Pneu- 
monic Sputum. — Talamon,  in  1883,  demonstrated  the  presence  of  this 
micrococcus  in  pneumonic  sputum,  described  its  morphological  char- 
acters, and  produced  typical  croupous  pneumonia  in  rabbits  by  in- 
jecting material  containing  it  into  the  lungs  through  the  thoracic 
walls. 

Salvioli,  in  1884,  demonstrated  its  presence  in  pneumonic  sputum 
by  injections  into  rabbits. 

In  1885  the  writer  made  a  similar  demonstration,  and  by  compara- 
tive experiments  showed  that  the  micrococcus  present  in  the  blood 
of  rabbits  inoculated  with  the  rusty  sputum  of  pneumonia  was  iden- 
tical with  that  which  he  had  discovered  in  1880  in  rabbits  inoculated 
with  his  own  saliva. 

The  same  year  (1885)  A.  Frankel  made  a  similar  demonstration, 
and  published  a  paper  containing  valuable  additions  to  our  knowl- 
edge relating  to  the  biological  characters  of  this  microorganism  (first 
publication  appeared  July  13th,  1885). 

In  1886  Weichselbaum  published  the  results  of  his  extended  re- 
searches relating  to  the  presence  of  this  micrococcus  in  the  fibrinous 
exudate  of  croupous  pneumonia.  He  obtained  it  in  ninety-four  cases 
(fifty-four  times  in  cultures)  out  of  one  hundred  and  twenty-nine  cases 
examined. 

Wolf  (1887)  found  it  in  sixty-six  cases  out  of  seventy  examined. 

Netter  (1887)  in  seventy-five  per  cent  of  his  cases,  and  in  the  sputum 
of  convalescents  from  pneumonia  in  sixty  per  cent  of  the  cases  ex- 
amined, by  inoculations  into  rabbits. 

Gameleia  (1887)  in  twelve  fatal  cases  of  pneumonia  in  which  he 
collected  material  from  the  lungs  at  the  post-mortem  examination. 

Goldenberg,  whose  researches  were  made  in  Gameleia's  labora- 
tory, found  it  in  pneumonic  sputum  in  forty  consecutive  cases,  by 
inoculations  into  rabbits  and  mice. 

The  Presence  of  Micrococcus  Pneumonice  Crouposce  in  Menin- 
gitis.— Numerous  bacteriologists  have  reported  finding  diplococci  in 
the  pus  of  meningitis,  and  frequently  the  microorganisms  have  been 
fully  identified  as  "  diplococcuspneumonise."  Thus  Netter  (1889),  in 
a  resume  of  the  results  of  researches  made  by  him  in  twenty-five 
cases  of  purulent  meningitis,  reports  as  follows  : 


BACTERIA  IN  CROUPOUS   PNEUMONIA.  401 

Thirteen  cases  were  examined  microscopically,  by  cultures,  and 
by  inoculations  into  susceptible  animals  ;  six  cases  by  microscopical 
examination  and  experiments  on  animals;  and  the  remainder  only  by 
microscopical  examination.  Four  of  the  cases  were  complicated 
with  purulent  otitis,  six  with  pneumonia,  three  with  ulcerative  endo- 
carditis. The  "  pneumococcus  "  was  found  in  sixteen  of  the  twenty- 
five  cases ;  in  four  Streptococcus  pyogenes  was  present ;  in  two 
Diplococcus  intracellularis  meningitidis  of  Weichselbaum ;  in  one 
Friedlander's  bacillus  ;  in  one  Newmann  and  Schaffer's  motile  ba- 
cillus ;  in  one  a  small  curved  bacillus. 

In  forty-five  cases  collected  from  the  literature  of  the  subject  by 


FIG.  90.  FIG.  91.  FIG.  98. 

FIG.  90.— Micrococcus  pneumonise  crouposae  from  blood  of  rabbit  inoculated  with  normal  human 
saliva  (Dr.  S.).  X  1,000. 

FIG.  91. — Micrococcus  pneumonias  crouposae  from  blood  of  rabbit  inoculated  subcutaneously 
with  fresh  pneumonic  sputum  from  a  patient  in  the  seventh  day  of  the  disease.  X  1,000. 

FIG.  92.— Surface  culture  of  Micrococcus  pneumonias  crouposse,  on  nutrient  agar,  showing  the 
development  of  long  chains.  X  1,000.1 

better  this  micrococcus  was  present  in  twenty-seven,  Streptococcus 
pyogenes  in  six,  and  the  Diplococcus  intracellularis  meningitidis  of 
Weichselbaum  in  ten. 

Monti  (1889),  in  four  cases  of  cerebro-spinal  meningitis,  demon- 
strated the  presence  of  the  same  micrococcus.  In  three  of  his  cases 
pneumonia  was  also  present.  In  two  Staphylococcus  pyogenes  aureus 
was  associated  with  the  "  diplococcus  pneumonia." 

Micrococcus  Pneumonias  Crouposce  in  Ulcerative  Endocar- 
ditis.— Weichselbaum,  in  a  series  of  twenty-nine  cases  examined 
(1888),  found  "  diplococcus  pneumonise"  in  seven. 

Micrococcus  Pneumonice  Crouposce  in  Acute  Abscesses. — In  a 
case  of  parotitis  occurring  as  a  complication  of  croupous  pneumonia 
this  micrococcus  was  obtained  from  the  pus  in  pure  cultures  by  Testi 
(1889);  and  in  another  case  in  which,  as  a  complication  of  pneumonia, 
there  developed  a  purulent  pleuritis,  abscess  of  the  parotid  on  both 
sides,  and  multiple  subcutaneous  abscesses,  the  pus  from  all  of  the 
sources  named  contained  the  ' '  diplococcus  "  in  great  numbers,  as 
shown  not  only  by  microscopical  examination  but  by  inoculation  into 
rabbits. 

1  The  above  figures  are  from  Dr.  Sternberg's  paper  published  in  the  American 
Journal  of  the  Medical  Sciences  for  July  and  October,  1885. 
26 


402  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

In  a  case  of  tonsillitis  resulting  in  the  formation  of  an  abscess 
Gabbi  (1889)  obtained  the  same  coccus  in  pure  cultures. 

In  otitis  media  this  micrococcus  has  been  found  in  a  consider- 
able number  of  cases  in  the  pus  obtained  by  paracentesis  of  the 
tympanic  membrane,  and  quite  frequently  in  pure  cultures — by  Zau- 
fal  (1889)  in  six  cases;  Levy  and  Schrader  (1889)  in  three  out  of  ten 
cases  in  which  paracentesis  was  performed;  by  Netter  (1889)  in  five 
out  of  eighteen  cases  occurring  in  children. 

Monti  (1889)  and  Belfanti  (1889)  report  cases  of  arthritis  of  the 
wrist  joint,  occurring  as  a  complication  of  pneumonia,  in  which  this 
micrococcus  was  obtained  in  pure  cultures.  Ortmann  and  Samter 
(1889),  in  a  case  of  purulent  inflammation  of  the  shoulder  joint  fol- 
lowing pneumonia  and  pleurisy,  obtained  the  "diplococcus  pneu- 
monise  "  in  pure  cultures. 

Morphology. — Spherical  or  oval  cocci,  usually  united  in  pairs,  or 
in  chains  consisting  of  three  or  four  elements.  Longer  chains,  con- 
taining ten  or  more  elements,  are  frequently  formed,  especially  in 
cultures  upon  the  surface  of  nutrient  agar,  and  in  liquid  media;  it 
may  therefore  be  regarded  as  a  streptococcus.  As  observed  in  the 
blood  of  inoculated  animals  it  is  usually  in  pairs  consisting  of  oval 
or  lance-oval  elements,  which  are  surrounded  by  a  transparent  cap- 
sule. Owing  to  the  elongated  form  of  the  cocci  when  in  active 
growth,  it  has  been  regarded  by  some  authors  as  a  bacillus;  but  in 
cultures  in  liquid  media,  when  development  by  binary  division  has 
ceased,  the  cells  are  spherical,  or  nearly  so,  and  in  cultures  on  the 
surface  of  nutrient  agar  the  individual  cells  more  nearly  approach  a 
spherical  form  than  in  the  blood  of  an  inoculated  animal.  The  "  lan- 
ceolate "  form  was  first  referred  to  by  Tala- 
mon,  who  described  it  as  having  the  form  of 
a  grain  of  wheat,  or  even  still  more  elongated 
like  a  grain  of  barley,  as  seen  in  the  fibrin- 
ous  exudate  of  croupous  pneumonia.  The 
transparent  material  surrounding  the  cells- 
so-called  capsule — is  best  seen  in  stained 
preparations  from  the  fibrinous  exudate  of 
FIG.  93.-Micrococcus  pneu-  croupous  pneumonia  or  from  the  blood  of  an 

monise  crouposae,  showing  cap- 
sule, attached  to  pus  cells  from     inoculated    animal.       It    appears    as    an  un- 

st«ined  marginal  band  surrounding  the  ellip- 
tical  cells,  and  varies  greatly  as  to  its  extent 
in  different  preparations.  This  capsule  probably  consists  of  a  sub- 
stance resembling  mucin,  and,  being  soluble  in  water,  its  extent  de- 
pends partly  upon  the  methods  employed  in  preparing  specimens  for 
microscopical  examination.  It  is  occasionally  seen  in  stained  prep- 
arations from  the  surface  of  cultures  on  blood  serum ;  and  in  drop 


BACTERIA  IN   CROUPOUS   PNEUMONIA.  403 

cultures  examined  under  the  microscope,  by  using  a  small  diaphragm 
it  may  be  seen  to  surround  the  cocci  as  a  scarcely  visible  halo. 

This  micrococcus  stains  readily  with  the  aniline  colors  ;  and  also 
by  Gram's  method,  which  constitutes  an  important  character  for  dis- 
tinguishing it  from  Friedlander's  bacillus. 

Biological  Characters.  —  Grows  in  the  presence  of  oxygen  — 
aerobic  —  but  is  also  a  facultative  anaerobic.  Like  other  micro- 
cocci,  it  has  no  spontaneous  movements.  It  grows  in  a  variety  of 
culture  media  when  they  have  a  slightly  alkaline  reaction,  but  will 
not  develop  in  a  medium  which  contains  the  slightest  trace  of  free 
acid.  Nor  will  it  grow  at  the  ordinary  room  temperature.  Scanty 
development  may  occur  at  a  temperature  of  22°  to  24°  C.,  but  a 
temperature  of  35°  to  37°  C.  is  most  favorable  for  its  growth,  which 
is  very  rapid  in  a  suitable  liquid  medium.  In  an  infusion  made  from 
the  flesh  of  a  chicken  or  a  rabbit  it  multiplies,  in  the  incubating 
oven,  with  remarkable  rapidity  ;  at  the  end  of  six  to  twelve  hours 
after  inoculation  the  previously  transparent  fluid  will  be  found  to 
present  a  slight  cloudiness  and  to  be  filled  throughout  with  the  cocci 
in  pairs  and  short  chains.  It  does  not  produce  a  milky  opacity  in 
liquid  media,  like  the  pus  cocci,  for  example,  but  the  fluid  becomes 
slightly  clouded  ;  multiplication  ceases  at  the  end  of  about  forty- 
eight  hours  or  less,  and  the  liquid  medium  again  becomes  transpa- 
rent as  a  result  of  the  subsidence  of  the  cocci  to  the  bottom  of  the 
receptacle. 

It  may  be  cultivated  in  flesh-peptone-gelatin,  containing  fifteen 
per  cent  of  gelatin,  at  a  temperature  of  24°  C.,  or  in  liquefied  gela- 
tin (ten  per  cent)  in  the  incubating  oven. 
In  gelatin  (fifteen  per  cent)  stab  cultures 
small  white  colonies  develop  all  along  the 
line  of  puncture,  and  in  gelatin  plates 
small,  spherical,  slightly  granular,  whitish 
colonies  are  formed  :  the  gelatin  is  not 
liquefied.  In  agar  plates  extremely  mi- 
nute colonies  are  developed  in  the  course 
of  forty-eight  hours,  which  resemble  little, 
transparent  drops  of  fluid,  and  under  the 
microscope  some  of  these  are  observed  to  FIG.  94 

have    a    Compact,    finely    granular    Central     coccus  pneumonias  crouposae  upon 

portion  surrounded  by  a  paler,  transparent,    XFSSSSSSSiS*  °' 

finely  granular  marginal  zone.     Upon  the 

surface  of  nutrient  agar  or  coagulated  blood  serum  development 
occurs  in  the  form  of  minute,  transparent,  jelly-like  drops,  which 
form  a  thin  layer  along  the  line  of  inoculation  in  "streak  cultures"  ; 
and  in  agar  stick  cultures  the  growth  along  the  line  of  puncture  is 


icro- 


404  BACTERIA   IN  CROUPOUS   PNEUMONIA. 

rather  scanty,  almost  homogeneous,  and  semi-transparent.  Upon 
potato  no  development  occurs,  even  in  the  incubating  oven.  Milk  is 
a  favorable  culture  medium,  and  the  casein  is  coagulated  as  a  result 
of  its  presence. 

It  ceases  to  grow  on  solid  media  at  about  40°  C.,  and  in  favorable 
liquid  media  at  42°  C.  Its  thermal  death-point,  as  determined  by 
the  writer,  is  52°  C. ,  the  time  of  exposure  being  ten  minutes.  It 
loses  its  vitality  in  cultures  in  a  comparatively  short  time — four  or 
five  days  on  agar — and  is  very  sensitive  to  the  action  of  germicidal 
agents.  Its  pathogenic  power  also  undergoes  attenuation  very 
quickly  when  it  is  cultivated  in  artificial  media,  but  may  be  restored 
by  passing  it  through  the  bodies  of  susceptible  animals.  Attenua- 
tion of  virulence  may  also  be  effected  by  exposing  bouillon  cultures 
to  a  temperature  of  42°  C.  for  twenty-four  hours,  or  by  five  days' 
exposure  to  a  temperature  of  41°  C. 

Emmerich  reported  in  1891  to  the  Congress  of  Hygiene  and 
Demography  in  London  the  results  of  experiments  made  by  him 
relating  to  immunity  in  rabbits  and  mice.  Rabbits  were  rendered 
immune  by  the  intravenous  injection  of  a  very  much  diluted  but 
virulent  culture  of  the  micrococcus.  The  flesh  of  these  immune 
rabbits  was  rubbed  up  into  a  fine  paste,  and  the  juices  obtained  by 
compressing  it  in  a  clean,  sterilized  cloth.  This  bloody  juice  was  kept 
for  twelve  hours  at  a  temperature  of  10°  C.,  and  then  sterilized  by 
passing  it  through  a  Pasteur  filter.  Some  of  this  juice  was  injected 
into  a  rabbit,  which  with  twenty-five  others  was  then  made  to  re- 
spire an  atmosphere  charged  with  a  spray  of  a  bouillon  culture  of 
the  micrococcus.  As  a  result  of  this  all  of  the  rabbits  died  except  the 
one  which  had  previously  been  injected  with  the  immunizing  juice. 
In  a  similar  experiment  upon  mice  six  of  these  animals,  which  had 
previously  been  injected  with  the  immunizing  juice,  survived  the  in- 
jection of  a  full  dose  of  a  virulent  culture,  while  a  control  mouse, 
not  previously  injected  with  the  juice,  promptly  died  after  receiving 
the  same  quantity  of  the  virulent  culture. 

The  writer  in  1881,  in  experiments  made  to  determine  the  value 
of  various  disinfectants,  as  tested  upon  this  micrococcus,  obtained 
experimental  evidence  that  its  virulence  is  attenuated  by  the  action 
of  certain  antiseptic  agents.  Commenting  upon  the  results  of  these 
experiments  in  my  chapter  on  "  Attenuation  of  Virus,"  in  "  Bacte- 
ria "  (1884),  I  say  : 

' '  Sodium  hyposulphite  and  alcohol  were  the  chemical  reagents  which 
produced  the  result  noted  in  these  experiments  ;  but  it  seems  probable  that 
a  variety  of  antiseptic  substances  will  be  found  to  be  equally  effective  when 
used  in  the  proper  proportion.  Subsequent  experiments  have  shown  that 
neither  of  these  agents  is  capable  of  destroying  the  vitality  of  this  septic 
micrococcus  in  the  proportion  used  (one  per  cent  of  sodium  hyposulphite  or 


BACTERIA   IN   CROUPOUS   PNEUMONIA.  405 

one  part  of  ninety-five-per-ceiit  alcohol  to  three  parts  of  virus),  and  that 
both  have  a  restraining-  influence  upon  the  development  of  this  microorgan- 
ism in  culture  fluids." 

The  following  results  were  obtained  by  the  writer  in  his  experi- 
ments (1881  and  1883)  to  determine  the  germicidal  and  antiseptic 
value  of  the  agents  named,  as  tested  upon  this  micrococcus. 

Alcohol. — A  twenty-four-per-cent  solution  was  effective  upon 
bouillon  cultures  in  two  hours. 

Boric  Acid. — A  saturated  solution  failed  to  destroy  vitality  after 
two  hours'  exposure,  but  1  :  400  restrained  development. 

Carbolic  Acid. — A  one-per-cent  solution  destroys  vitality  in  two 
hours,  and  1  :  500  restrains  development. 

Cupric  Sulphate  destroys  the  virulence  of  the  coccus  in  the 
blood  of  a  rabbit  in  the  proportion  of  1  :  400  in  half  an  hour. 

Ferric  Sulphate  failed  to  destroy  vitality  in  a  saturated  solution, 
but  restrained  development  in  the  proportion  of  1  :  200. 

Hydrochloric  Acid  destroys  the  virulence  of  the  blood  of  a  rab- 
bit containing  this  micrococcus  in  the  proportion  of  1  :  200. 

Iodine,  in  aqueous  solution  with  potassium  iodide,  destroys  vital- 
ity in  the  proportion  of  1: 1,000  and  prevents  development  in  1:  4,000. 

Mercuric  Chloride. — One  part  in  forty  thousand  prevents  the 
development  of  this  micrococcus,  and  1  :  20,000  was  found  to  destroy 
vitality  in  two  hours. 

Nitric  Acid. — One  part  in  four  hundred  destroyed  the  virulence 
of  rabbit's  blood  containing  this  micrococcus. 

Caustic  Potash. — A  two-per-cent  solution  destroyed  vitality  in 
two  hours. 

Potassium  Permanganate. — A  two-per-cent  solution  destroyed 
the  virulence  of  rabbit's  blood  containing  this  coccus. 

Salicylic  Acid,  dissolved  by  the  addition  of  sodium  biborate. — 
A  solution  of  1  :  400  prevented  development. 

Sulphuric  Acid. — One  part  in  two  hundred  destroys  vitality,  and 
1  :  800  prevents  development. 

In  a  paper  by  Bordoni-Uffreduzzi  relating  to  the  resisting  power 
of  pneumonic  virus  for  desiccation  and  light,  the  following  results  are 
given :  Pneumonic  sputum  attached  to  cloths,  when  dried  in  the  air 
and  exposed  to  diffuse  daylight,  retained  its  virulence,  as  shown  by 
injection  in  rabbits,  for  a  period  of  nineteen  days  in  one  series  of  ex- 
periments and  for  fifty-five  days  in  another.  Exposed  to  direct  sun- 
light the  same  material  retained  its  virulence  after  twelve  hours' 
exposure.  Cultures  have  far  less  resistance,  and  the  protection 
afforded  by  the  dried  albuminous  material  in  which  the  micrococci 
were  embedded,  in  the  experiments  referred  to,  probably  accounts 
for  the  virulence  being  retained  so  long  a  time. 


406  BACTERIA  IN  CROUPOUS   PNEUMONIA. 

Kruse  and  Pansini  (1892)  have  published  an  elaborate  paper  giv- 
ing an  account  of  their  researches  relating  to  "diplococcus  pneumo- 
nia "  and  allied  streptococci.  We  give  below  a  summary  statement 
of  their  results : 

Many  varieties  were  obtained  by  the  observers  named  in  their  cultures 
from  various  sources — from  the  lungs  of  individuals  dead  from  pneumonia, 
from  pleuritic  exudate,  from  pneumonic  sputa,  from  bronchitic  sputa,  from 
the  saliva  of  healthy  persons,  from  the  secretion  in  a  case  of  subacute  nasal 
catarrh,  from  the  urine  of  a  patient  with  nephritis. 

Pure  cultures  were  obtained  by  the  use  of  agar  plates  or  by  inoculations 
into  rabbits.  In  all  about  thirty  varieties  were  obtained  and  cultivated 
through  many  successive  generations.  As  a  rule,  the  different  varieties, 
which  at  first  were  seen  to  nave  the  form  of  diplococci,  when  cultivated  for 
a  length  of  time  in  artificial  media  presented  the  form  of  streptococci  ;  and 
the  elements  which  at  first  were  lancet-shaped  showed  a  tendency  to  become 
spherical. 

The  more  virulent  varieties  usually  presented  the  form  of  diplococci 
with  lancet-shaped  elements,  or  of  short  chains.  A  variety  which  formed 
long  chains  could  be  pronounced,  in  advance  of  the  experiments  on  animals, 
to  possess  comparatively  little  virulence.  When  by  inoculations  in  animals 
the  virulence  of  such  a  variety  was  restored,  the  tendency  to  form  chains 
was  less  pronounced. 

Although,  as  a  rule,  no  development  occurs  at  20°  C.,  certain  varieties 
were  obtained  which,  after  long  cultivation  in  artificial  media,  showed  a  de- 
cided growth  at  18°  C. 

Decided  differences  were  shown  by  the  cultures  from  various  sources  as 
regards  their  growth  in  milk.  Out  of  eighty-four  cultures  from  various 
sources  eleven  did  not  produce  coagulation.  As  a  rule,  cultures  which  caused 
coagulation  of  milk  were  virulent  for  rabbits,  and  when  such  cultures  lost 
their  virulence  they  usually  lost  at  the  same  time  the  power  of  coagulating 
milk.  Virulent  cultures  die  out  sooner  than  those  which  have  become  at- 
tenuated by  continuous  cultivation  in  artificial  media;  the  first,  on  the  sur- 
face of  agar,  usually  fail  to  grow  at  the  end  of  a  week,  while  the  attenuated 
cultures  may  survive  for  three  weeks  or  more. 

Pathogenesis. — This  micrococcus  is  very  pathogenic  for  mice  and 
for  rabbits,  less  so  for  guinea-pigs.  The  injection  of  a  minute  quan- 
tity— 0. 2  cubic  centimetre  or  less — of  a  virulent  culture  beneath  the 
skin  of  a  rabbit  or  a  mouse  usually  results  in  the  death  of  the  animal 
in  from  twenty-four  to  forty-eight  hours.  The  following  is  from  the 
writer's  first  published  paper  (1881),  and  refers  to  the  pathological 
appearances  in  rabbits  : 

"  The  course  of  the  disease  and  the  post-mortem  appearances  indicate  that 
it  is  a  form  of  septicaemia.  Immediately  after  the  injection  there  is  a  rise  of 
temperature,  which  in  a  few  hours  may  reach  2°  to  3°  C.  (3.6°  to  5.4°  F.) ; 
the  temperature  subsequently  falls,  and  shortly  before  death  is  often  several 
degrees  below  the  normal.  There  is  loss  of  appetite  and  marked  debility 
after  twenty -four  hours,  and  the  animal  commonly  dies  during  the  second 
night  or  early  in  the  morning  of  the  second  day  after  the  injection.  Death 
occurs  still  more  quickly  when  the  blood  from  a  rabbit  recently  dead  is  in- 
jected. Not  infrequently  convulsions  immediately  precede  death. 

"The  most  marked  pathological  appearance  is  a  diffuse  inflammatory 
cedema  or  cellulitis,  extending  in  all  directions  from  the  point  of  injection, 


BACTERIA   IN  CROUPOUS  PNEUMONIA. 


407 


but  especially  to  the  dependent  portions  of  the  body.  Occasionally  there  is 
a  little  pus  near  the  puncture,  but  usually  death  occurs  before  the'cellulitis 
reaches  the  point  of  producing  pus.  The  subcutaneous  connective  tissue 
contains  a  quantity  of  bloody  serum,  which  possesses  virulent  properties  and 
which  contains  a  multitude  of  micrococci.  There  is  usuc  lly  more  or  less  in- 
flammatory adhesion  of  the  integument  to  the  subjacent  tissues.  The  liver 
is  sometimes  dark-colored  and  gorged  with  blood,  but  more  frequently  it  is 
of  a  lighter  color  than  normal  and  contains  much  fat.  The  spleen  is  either 
normal  in  appearance  or  enlarged  and  dark-colored.  Changes  in  this  organ 
are  more  marked  in  those  cases  which  are  of  the  longest  duration. 

' '  The  blood  commonly  contains  an  immense  number  of  micrococci,  usually 
joined  in  pairs  and  having  a  diameter  of  about  0.5  /*.  These  are  found  in 
blood  drawn  from  superficial  veins,  from  arteries,  and  from  the  cavities  of 
the  heart  immediately  after  death,  and  in  a  few  cases  their  presence  has  been 


FIG.  93. — Micrococcus  pneumonias  crouposae  in  blood  of  rabbit  inoculated  with  pneumonic  spu- 
tum.    X  1,000. 

verified  during  life.  Observations  thus  far  made,  however,  indicate  that  it 
is  only  during  the  last  hours  of  life  that  these  parasites  multiply  in  the  cir- 
culating fluid,  and  in  a  certain  proportion  of  the  cases  a  careful  search  has 
failed  to  reveal  their  presence  in  the  blood  in  post-mortem  examinations 
made  immediately  after  the  death  of  the  animal." 

In  animals  which  are  not  examined  until  some  hours  after  death 
a  considerable  increase  in  the  number  of  micrococci  occurs  post  mor- 
tem. The  fact  that  this  micrococcus  varies  very  much  as  to  its 
pathogenic  power,  as  a  result  of  conditions  relating  to  the  medium  in 
which  it  develops,  was  insisted  upon  in  my  first  published  paper,  and 
has  been  fully  established  by  later  researches  (Frankel,  Gameleia). 
Susceptible  animals  inoculated  with  attenuated  cultures  acquire  an 
immunity  against  virulent  cultures. 

In  dogs  subcutaneous  injections  usually  give  a  negative  result, 
or  at  most  a  small  abscess  forms  at  the  point  of  inoculation.  In  a 


408  BACTERIA   IN   CROUPOUS   PNEUMONIA. 

single  experiment,  however,  the  writer  has  seen  a  fatal  result  in  a 
dog  from  the  injection  of  one  cubic  centimetre  of  bloody  serum  from 
the  subcutaneous  connective  tissue  of  a  rabbit  recently  dead.  This 
shows  the  intense  virulence  of  the  micrococcus  when  cultivated  in 
the  body  of  this  animal.  Pneumonia  never  results  from  subcutane- 
ous injections  into  susceptible  animals,  but  injections  made  through 
the  thoracic  walls  into  the  substance  of  the  lung  may  induce  a  typi- 
cal fibrinous  pneumonia.  This  was  first  demonstrated  by  Talamon 
(1883),  who  injected  the  fibrinous  exudate  of  croupous  pneumonia, 
obtained  after  death,  or  drawn  during  life  by  means  of  a  Pravaz 
syringe  from  the  hepatized  portions  of  the  lung,  into  the  lungs  of 
rabbits.  According  to  See,  eight  out  of  twenty  animals  experi- 
mented upon  exhibited  "a  veritable  lobar,  fibrinous  pneumonia, 
with  pleurisy  and  pericarditis  of  the  same  nature."  Gameleia  has 
also  induced  pneumonia  in  a  large  number  of  rabbits,  and  also  in  the 
dog  and  the  sheep,  by  injections  directly  into  the  pulmonary  tissue. 
Sheep  were  found  to  survive  subcutaneous  inoculations,  unless  very 
large  doses  (five  cubic  centimetres)  of  the  most  potent  virus  were  in- 
jected. But  intrapulmonary  inoculations  invariably  induced  a  typi- 
cal fibrinous  pneumonia  which  usually  proved  fatal.  In  dogs  simi- 
lar injections  gave  rise  to  a  "  frank,  fibrinous  pneumonia  which 
rarely  proved  fatal,  recovery  usually  occurring  in  from  ten  to  fifteen 
days,  after  the  animal  had  passed  through  the  stages  of  red  and 
gray  hepatization  characteristic  of  this  affection  in  man." 

Monti  claims  to  have  produced  typical  pneumonia  in  rabbits  by 
injecting  cultures  of  this  micrococcus  into  the  trachea. 

From  the  evidence  obtained  in  these  experimental  inoculations, 
and  that  recorded  relating  to  the  presence  of  this  micrococcus  in  the 
fibrinous  exudate  of  croupous  pneumonia,  we  are  justified  in  con- 
cluding that  it  is  the  usual  cause  of  this  disease,  and  consequently 
have  described  it  under  the  name  Micrococcous  pneumonias  crou- 
posae.  We  prefer  this  to  the  name  commonly  employed  by  German 
authors — "  diplococcus  pneumonias  "  —because  this  micrococcus,  al- 
though commonly  seen  in  pairs,  forms  numerous  short  chains  of 
three  or  four  elements  in  cultures  in  liquid  media,  and  upon  the  sur- 
face of  nutrient  agar  may  grow  out  into  long  chains.  It  would, 
therefore,  more  properly  be  called  a  streptococcus  than  a  diplococcus. 

While  the  micrococcus  of  pneumonia  is  not  usually  seen  in  the 
blood  in  cases  of  pneumonia  it  is  probably  present  in  small  numbers, 
and  secondary  infection  of  the  kidneys  appears  to  be  a  common  occur- 
rence. Thus  Frankel  and  Reiche  (1894)  report  that  in  twenty-two 
cases  out  of  twenty-four  in  which  they  had  an  opportunity  to  exam- 
ine the  kidneys,  this  micrococcus  was  present.  It  was  found  espe- 


BACTERIA   IN  CROUPOUS  PNEUMONIA.  409 

cially  in  the  larger  branches  of  the  veins  and  arteries,  but  also  in  the 
intertubular  vessels  and  the  glomeruli.  The  kidneys  gave  evidence 
of  degenerative  changes,  and  it  is  probable  that  the  "  pneumococcus  " 
would  have  been  found  in  the  urine  of  some  of  these  cases  if  a  bac- 
teriological examination  had  been  made  during  life. 


VII. 

PATHOGENIC  MICROCOCCI   NOT   DESCRIBED   IN 
SECTIONS  V.  AND  VI. 

DIPLOCOCCUS  INTRACELLULARIS   MENINGITIDIS. 

DISCOVERED  by  Weichselbaum  (1887)  in  the  exudate  of  cerebro 
spinal  meningitis  (six  cases),  for  the  most  part  within  the  cells. 

Morphology. — Micrococci,  usually  united  in  pairs,  in  groups  of 
four,  or  in  little  masses ;  sometimes  solitary  and  larger  (probably 
being  upon  the  point  of  dividing).  Distinguished  by  their  presence 
in  the  interior  of  pus  cells  in  the  exudate,  in  this  respect  resembling 
the  gonococcus. 

Stain  best  with  Loffler's  alkaline  solution  of  methylene  blue. 
Do  not  retain  their  color  when  treated  with  iodine  solution  (Gram's 
method). 

Biological  Characters. — This  micrococcus  does  not  grow  at  the 
room  temperature,  but  upon  nutrient  agar  an  abundant  development 
occurs  in  the  incubating  oven.  Upon  the  surface  of  agar  a  tolerably 
luxuriant,  viscid  growth,  which  by  reflected  light  is  gray  and  by 
transmitted  light  grayish- white  ;  along  the  line  of  puncture  growth 
occurs  only  near  the  surface,  indicating  that  this  micrococcus  will 
not  grow  in  the  absence  of  oxygen.  Upon  plates  made  from  agar- 
agar  (one  per  cent)  and  gelatin  (two  per  cent)  very  small  colonies  are 
formed  in  the  interior  of  the  mass,  and  larger  ones,  of  a  grayish 
color,  on  the  surface.  The  former,  under  the  microscope,  are  seen  to 
be  round  or  slightly  irregular,  finely  granular,  and  of  a  yellowish- 
brown  color.  The  superficial  colonies  have  a  yellowish-brown  nu- 
cleus, surrounded  by  a  more  transparent  zone.  The  growth  upon 
coagulated  blood  serum  is  very  scanty,  as  is  that  in  bouillon ;  no 
growth  occurs  upon  potato.  This  micrococcus  quickly  loses  its  power 
of  reproduction  in  artificial  cultures — within  six  days — and  should 
be  transplanted  to  fresh  material  at  short  intervals — two  days. 

Pathogenesis. — Mice  are  especially  susceptible,  and  usually  die 
within  forty-eight  hours  after  inoculation.  Also  pathogenic  for 
guinea-pigs,  rabbits,  and  dogs. 


NOT   DESCRIBED   IN  SECTIONS  V.    AND   VI.  411 

MICROCOCCUS   TETRAGENUS. 

First  described  by  Gaffky  (Fliigge).  Obtained  by  Koch  and 
Gaffky  (1881)  from  a  cavity  in  the  lung  in  a  case  of  pulmonary 
phthisis.  Since  found  occasionally  in  normal  saliva  (three  times  in 
fifty  persons  examined  by  Biondi),  and  in  the  pus  of  acute  abscesses 
(Steinhaus,  Park,  Yangel).  Rather  common  in  the  sputum  of  phthi- 
sical cases. 

Morphology. — Micrococci,  having  a  diameter  of  about  one  /*, 
which  divide  in  two  directions,  forming  tetrads,  which  are  enclosed 
in  a  transparent,  jelly-like  envelope — especially  well  developed  as 
seen  in  the  blood  and  tissues  of  inoculated  animals.  In  cultures  the 
cocci  are  seen  in  the  various  stages  of  division,  as  large  single  cells, 


syvas 

m 

Fia.  96.— Micrococcus  tetragenus;  section  of  lung  of  mouse,    x  800.    (Flugge.) 

pairs  of  oval  elements,  or  groups  of  four  resulting  from  the  trans- 
verse division  of  these  latter. 

Stains  quickly  with  aniline  colors,  and  in  preparations  from  the 
blood  of  an  inoculated  animal  the  transparent  envelope  may  also  be 
feebly  stained.  Stains  also  by  Gram's  method. 

Biological  Characters. — This  micrococcus  grows,  rather  slowly, 
in  nutrient  gelatin  at  the  ordinary  room  temperature,  without  lique- 
faction of  the  gelatin.  Upon  gelatin  plates  small  white  colonies  are 
developed  in  from  twenty-four  to  forty-eight  hours,  which  under  the 
microscope,  with  a  low  power,  are  seen  to  be  spherical  or  lemon- 
shaped,  finely  granular,  and  with  a  mulberry -like  surface.  When 
they  come  to  the  surface  they  form  white,  elevated,  and  rather  thick 
masses  having  a  diameter  of  one  to  two  millimetres.  In  gelatin 
stab  cultures  a  broad  and  thick  white  mass  forms  upon  the  surface, 


412  PATHOGENIC   MICROCOCCI 

and  along  the  line  of  puncture  a  series  of  round,  milk-white  or  yel- 
lowish masses  form,  which  usually  remain  distinct,  but  may  become 
confluent.  Upon  the  surface  of  agar  the  growth  is  similar  to  that 
upon  gelatin,  or  in  streak  inoculations  may  consist  of  a  series  of 
spherical,  white  colonies.  Upon  cooked  potato  a  thick,  viscous  layer 
is  formed  of  milk-white  color  ;  the  growth  upon  blood  serum  is  also 
abundant,  especially  in  the  incubating  oven.  This  micrococcus  is  a 
facultative  anaerobic. 

Pathogenesis. — Subcutaneous  inoculation  of  a  culture  of  this 
micrococcus  in  minute  quantity  is  fatal  to  white  mice  in  from  two  to 
six  days.  The  animals  remain  apparently  well  for  the  first  day  or 
two,  then  remain  quiet  and  somnolent  until  death  occurs.  The  cocci 
are  found  in  comparatively  small  numbers  in  the  blood  of  the  heart, 
but  are  more  numerous  in  the  spleen,  lungs,  liver,  and  kidneys,  from 
which  organs  beautiful  stained  preparations  may  be  made  show- 
ing the  tetrads  surrounded  by  their  transparent  capsule.  Common 
house  mice  and  field  mice  are,  for  the  most  part,  immune,  as  are  the 
rabbit  and  the  dog.  Guinea-pigs  sometimes  die  from  general  infec- 
tion, and  sometimes  a  local  abscess  is  the  only  result  of  a  subcutane- 
ous inoculation. 

MICROCOCCUS  BOTRYOGENUS  (Rabe). 

Synonyms. — Micrococcus  of  "  myko-desmoids  "  of  the  horse;  Mi- 
crococcus askoformans  (Johne)  ;  Ascococcus  Johnei  (Cohn). 

First  described  by  Bollinger  (1870)  ;  morphological  characters  and 
location  in  the  diseased  tissues  described  by  Johne  (1884)  ;  biological 
characters  determined  by  Rabe  (1886). 

Is  found  in  certain  diffused  or  circumscribed  growths  in  the  con- 
nective tissue  of  horses — "  myko-desmoids." 

Morphology. — Micrococci,  having  a  diameter  of  1  to  1.5  /*,  usu- 
ally united  in  pairs. 

In  the  tissues  the  cocci  are  united  in  colonies  of  fifty  to  one  hun- 
dred yw  in  diameter,  and  these  are  associated  in  mulberry-like  masses 
visible  to  the  naked  eye.  The  separate  colonies  are  enclosed  in  a 
homogeneous,  transparent  envelope — as  in  Ascococcus  Billrothii. 
This  is  not  the  case,  however,  in  cultures  in  artificial  media. 

Stains  with  the  aniline  colors. 

Biological  Characters. — In  gelatin  plate  cultures  spherical, 
sharply  defined,  silver-gray  colonies  are  developed  ;  later  these  have 
a  yellowish  color  and  a  metallic  lustre,  and  the  plate  presents  the  ap- 
pearance of  being  powdered  with  grains  of  pollen.  It  gives  off  a 
peculiar  fruit-like  odor,  reminding  one  of  the  odor  of  strawberries. 
In  gelatin  stab  cultures  growth  occurs  along  the  line  of  puncture  as 
a  pale  grayish- white  line,  which  later  becomes  milk-white ;  an  air 


NOT  DESCRIBED   IN  SECTIONS  V.    AND   VI.  413 

bubble  forms  near  the  surface  of  the  gelatin  ;  very  slight  liquefac- 
tion occurs  in  the  immediate  vicinity  of  the  line  of  growth,  and  after 
a  time  the  grayish-white  thread  sinks  into  an  irregular  mass,  lying 
at  the  bottom  of  the  puncture.  Upon  nutrient  agar  scarcely  any  de- 
velopment occurs.  Upon  potato  the  growth  is  abundant,  in  the  form 
of  a  pale-yellow,  circular  layer,  and  the  culture  gives  off  the  peculiar 
odor  above  described. 

Pathogenesis. — When  inoculated  into  guinea-pigs  general  infec- 
tion and  death  result.  In  sheep  and  goats  it  produces  a  local  in- 
flammatory oedema  and  sometimes  necrosis  of  the  tissues.  In  horses 
inoculated  subcutaneously  an  inflammatory  oedema  first  occurs,  fol- 
lowed at  the  end  of  from  four  to  six  weeks  by  the  development  of  new 
growths  in  the  connective  tissue,  resembling  the  tumors  found  in 
cases  of  the  disease  in  the  animal  from  which  the  micrococcus  in 
question  was  first  cultivated.  These  tumors  contain  characteristic 
mulberry-like  conglomerations  of  colonies  made  up  of  the  coccus. 

MICROCOCCUS  OF  MANFREDI. 

Synonym. — Micrococcus  of  progressive  granuloma  formation. 

Obtained  by  Manfredi  (1886)  from  the  sputum  of  two  cases  of 
croupous  pneumonia  following  measles. 

^[orphology. — Oval  micrococci,  having  a  diameter  of  0.6  to  1.0  /* 
and  from  1.0  to  1.5  /I  in  length  ;  usually  associated  in  pairs,  and  oc- 
casionally in  short  chains  containing  three  or  four  elements. 

Stains  with  the  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — Aerobic ;  does  not  liquefy  gelatin. 
Upon  gelatin  plates  forms  small,  spherical  colonies,  at  first  grayish- 
white,  which  spread  out  upon  the  surface  as  thin,  transparent  plates, 
which  by  transmitted  light  have  a  bluish,  by  reflected  light  a  pearl- 
gray  color.  Later  these  become  thicker  and  have  a  pearly  lustre. 
Under  the  microscope  (forty  to  fifty  diameters)  the  colonies  are  seen 
to  be  slightly  granular  and  the  margins  have  an  irregular  outline. 
In  gelatin  stab  cultures  a  scanty  growth  occurs  along  the  line  of 
puncture,  and  a  rather  thin  and  limited  growth  about  the  point  of 
inoculation.  Upon  blood  serum  a  thin,  greenish-yellow  layer,  which 
has  irregular  margins  and  a  slightly  granular,  shining  surface,  is 
developed.  The  growth  upon  potato,  at  37°  C.,  is  scanty,  and  con- 
sists of  a  very  thin,  moist  layer,  which  has  a  yellowish  color  and  io 
slightly  granular.  Growth  occurs  in  favorable  media — bouillon, 
gelatin — at  temperatures  of  18°  to  48°  C.,  but  ceases  at  a  temperature 
of  48°  to  50°  C. 

Pathogenesis. — Pathogenic  for  dogs,  rabbits,  guinea-pigs,  mice, 
and  birds.  In  mammals  the  principal  pathological  appearance  re- 
sulting from  infection  consists  in  the  formation  of  "  granulation  tu- 


414  PATHOGENIC   MICROCOCCI 

mors  "  in  the  parenchymatous  organs.  These  vary  in  size  from  that 
of  a  millet  seed  to  that  of  a  pea,  and  undergo  caseation.  They  con- 
tain the  micrococcus  and  are  infectious.  Mammals  die  in  from  nine 
to  fifteen  days  ;  birds  in  from  one  to  three  or  four,  and  without  the 
formation  of  the  characteristic  granuloma,  but  with  general  infec- 
tion of  the  blood.  Cultures  which  have  been  kept  for  several  months 
retain  their  pathogenic  power. 

MICROCOCCUS   OF  BOVINE   MASTITIS   (Kitt). 

Obtained  by  Kitt  (1885)  from  the  udder  of  cows  suffering  from  mastitis 
and  giving-  milk  mixed  with  pus. 

Morphology. — Micrococci,  having  a  diameter  of  0.2  to  0.5  >u,  solitary, 
united  in  pairs,  in  irregular  groups,  and  occasionally  in  chains. 

Stains  with  the  aniline  colors. 

Biological  Characters. — Does  not  liquefy  gelatin.  Upon  gelatin  plates 
forms  spherical,  translucent,  glistening  colonies,  the  size  of  a  hemp  seed  to 
that  of  a  pin's  head ;  in  gelatin  stab  cultures  a  nail-shaped  growth  occurs, 
the  mass  at  the  point  of  puncture  being  opaque  and  of  a  white  color.  Upon 
potato,  colonies  are  quickly  developed  which  have  a  grayish -white  or  dirty 
yellow  color,  and  after  a  few  days  have  a  shining,  wax-like  appearance. 
G-rows  rapidly  in  milk,  causing  an  acid  reaction ;  in  six  hours  in  the  incu- 
bating oven  the  milk  is  pervaded  by  the  micrococcus,  or  in  twelve  hours  at 
20°  C. 

Pathogenesis. — Injection  of  pure  cultures,  suspended  in  distilled  water, 
into  the  mammary  glands  of  cows,  produces  typical,  acute,  purulent  mas- 
titis (Kitt).  The  micrococcus  produced  the  same  result  after  having  been 
cultivated  in  artificial  media  for  a  year.  Subcutaneous  inoculations  in  cows, 
pigs,  guinea-pigs,  rabbits,  and  mice  were  without  result.  Injections  into 
the  mammary  gland  of  goats  were  also  without  effect. 

MICROCOCCUS   OF   BOVINE   PNEUMONIA  (?). 

Isolated  by  Poels  and  Nolen  (1886)  from  the  lungs  of  cattle  suffering 
from  ' '  Lungenseuche  "  (infectious  pleuro-pneumonia  of  cattle). 

Morphology. — Micrococci,  varying  considerably  in  size — average  dia- 
meter 0.9  #;  solitary,  in  pairs,  or  in  chains  containing  several  elements;  sur- 
rounded by  a  transparent  capsule,  which  stains  with  difficulty. 

Stains  with  all  the  aniline  colors,  and  with  difficulty  by  Gram's  method. 

Biological  Characters. — Does  not  liquefy  gelatin,  and  grows  like  the  ba- 
cillus of  Friedlander  in  gelatin  stab  cultures  (nail-shaped  growth).  In  gela- 
tin plates  the  colonies  are  spherical,  white,  and  have  a  very  faint  yellowish 
tinge.  Grows  more  rapidly  on  agar  in  the  incubating  oven,  and  upon  po- 
tato in  the  form  of  a  very  pale-yellowish  layer.  Is  destroyed  by  a  tempera- 
ture of  66°  C.  maintained  for  fifteen  minutes. 

Pathogenesis  — Pure  cultures  injected  into  the  lungs  of  dogs,  rabbits, 
and  guinea-pigs  are  said  to  give  rise  to  pneumonic  inflammation,  and  simi- 
lar results  were  obtained  by  injection  into  the  trachea  of  dogs  and  by  in- 
halation experiments.  Injection  of  a  pure  culture  into  the  lungs  of  a  cow 
caused  extensive  pneumonic  changes ;  but  these  did  not  entirely  correspond 
with  the  appearances  found  in  the  lungs  of  cattle  suffering  from  infectious 
pneumonia.  Cattle  inoculated  with  a  pure  culture,  by  means  of  a  sterilized 
lancet,  did  not  fall  sick,  but  are  believed  by  Poels  and  Nolen  to  have  been 
protected  from  the  disease  by  such  inoculations. 

The  specific  relation  of  the  micrococcus  above  described  to  the  disease 
with  which  it  was  associated,  in  the  researches  of  the  authors  mentioned,  has 
not  been  established  by  subsequent  investigations. 


NOT  DESCRIBED  IN  SECTIONS  V.    AND  VI.  415 

STREPTOCOCCUS   SEPTICUS   (Flugge). 

Found  by  Nicolaier  and  by  Guameri  in  unclean  soil  during  researches 
made  in  Fliigge?s  laboratory  in  Gottingen. 

Morphology. — Cannot  be  distinguished  from  Streptococcus  pyogenes,  but 
does  not  so  constantly  form  chains,  being  found  in  the  tissues  of  inoculated 
animals,  for  the  most  part  in  pairs. 

Biological  Characters. — Grows  more  slowly  than  Streptococcus  pyogenes ; 
in  gelatin  plates  very  minute  colonies  first  appear  at  the  end  of  three  or  four 
days,  or  along  the  line  of  puncture  in  gelatin  stick  cultures  after  five  or  six 
days.  Does  not  liquefy  gelatin. 

Pathogenesis. — Is  very  pathogenic  for  mice  and  for  rabbits,  causing  death 
from  general  infection  in  two  or  three  days. 

STREPTOCOCCUS  BOMBYCIS. 

Synonym. — Microzyma  bombycis  (Bechamp). 

Found  in  the  bodies  of  infected  silkworms  suffering  from  la  flacherie 
(maladie  des  morts-plats).  Etiological  relation  established  by  Pasteur. 

Morphology. — Oval  cells,  not  exceeding  1.5  /*  in  diameter,  in  pairs  or  in 
chains. 

Biological  Characters. — Not  determined  with  precision. 

Pathogenesis. — The  infected  silkworm  ceases  to  eat,  becomes  weak,  and 
dies.  Its  body  is  soft  and  diffluent,  and  at  the  end  of  twenty-four  to  forty- 
eight  hours  is  filled  with  a  dark-brown  fluid  and  with  gas. 

NOSEMA  BOMBYCIS. 

Synonyms. — Micrococcus  ovatus;  Panhistophyton  ovatum. 

Found  in  the  blood  and  all  of  the  organs  of  silkworms  infected  with 
pebriiie  (Fleckenkrankheit). 

First  observed  by  Cornalia.     Etiological  relation  established  by  Pasteur. 

Morphology. — Shining,  oval  cells,  three  to  four  #  long  and  twoy"  broad; 
solitary,  in  pairs,  or  in  irregular  groups. 

Biological  Characters. — Not  determined  with  precision. 

Pathogenesis. — Dark  spots  appear  upon  the  skin  of  infected  silkworms, 
which  lose  their  appetite,  become  slender  and  feeble,  and  soon  die.  The 
oval  corpuscles  are  found  in  all  of  the  organs,  and  also  in  the  eggs  of 
butterflies  hatched  from  infected  larvae.  Some  authors  are  of  the  opinion 
that  the  oval  corpuscles  found  in  this  disease  do  not  belong  to  the  bacte- 
ria, but  to  an  entirely  different  class  of  microorganisms — the  Psorospermia 
(Metschnikoff). 

MICROCOCCUS   OF   HEYDENREICH. 

Synonyms. — Micrococcus  of  Biskra  button — Fr.  "  clou  de  Biskra  ";  Ger. 
"  Pendesche  Geschwur." 

Found  by  Heydenreich  (1888)  in  pus  and  serous  fluid  obtained  from  the 
tumors  and  ulcers  in  the  Oriental  skin  affection  known  as  Biskra  button. 

Morphology. — Diplococci,  from  0.86  to  1  /*  in  length,  surrounded  by  a 
capsule ;  sometimes  associated  to  form  tetrads. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  liquefying  micrococcus.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  In  gelatin  stick  cultures, 
at  20°  C.,  at  the  end  of  forty-eight  hours  growth  occurs  along  the  line  of 
puncture  in  the  form  of  small,  crowded  colonies,  which  produce  a  grayish- 
white  line ;  upon  the  surface  a  thin,  circular  layer  of  a  yellowish-white 
color  is  developed.  At  the  end  of  three  to  four  days  liquefaction  commences 
near  the  surface,  where  a  funnel  is  formed  which  extends  until  about  the 
fourteenth  day,  when  the  gelatin  is  completely  liquefied.  Upon  the  surface 


416  PATHOGENIC  MICROCOCCI 

of  agar,  at  37°  C.,  a  grayish- white  or  yellowish  layer  is  formed  at  the  end  of 
twenty-four  hours,  which  has  a  varnish-like  lustre.  Upon  potato,  at  30°  to 
35°  C.,  at  the  end  of  forty-eight  hours  a  white  or  yellow  layer  has  de- 
veloped. 

Pathogenesis. — According  to  Heydenreich,  inoculations  in  rabbits,  dogs, 
chickens,  horses,  and  sheep  cause  a  skin  affection  which  is  identical  with 
that  which  characterizes  Biskra  button  in  man.  When  rubbed  into  the 
healthy  skin  of  man  it  also  produces  the  development  of  abscesses. 

MICROCOCCUS  ENDOCARDITIDIS  RUGATUS  (Weichselbaum). 

Obtained  by  Weichselbaum  (1890)  from  the  affected  cardiac  valves  in  a 
fatal  case  of  ulcerative  endocarditis. 

Morphology. — Micrococci,  resembling  the  staphylococci  of  pus  in  dimen- 
sions and  mode  of  grouping;  solitary,  in  pairs,  in  groups  of  four,  or  in  ir- 
regular masses. 

Biological  Characters.- — An  aerobic  micrococcus.  Does  not  grow  at  the 
room  temperature.  Upon  agar  plates,  at  37°  C. ,  at  the  end  of  three  or  four 
days  the  superficial  colonies  consist  of  a  small,  brown,  central  mass  sur- 
rounded by  a  granular,  semi- transparent,  grayish  marginal  zone;  gradually 
they  attain  a  characteristic  wrinkled  appearance ;  the  deep  colonies,  under  a 
low  power,  are  irregular,  finely  granular,  and  contain  a  large  central,  yel- 
lowish-brown nucleus  surrounded  by  a  narrow,  grayish-brown  peripheral 
zone.  In  agar  stab  cultures  small,  spherical  colonies  are  formed  upon  the 
surface,  which  become  confluent,  forming  a  grayish-white,  wrinkled  layer 
which  has  a  stearin-like  lustre  and  is  very  viscid ;  a  scanty  growth  occurs 
along  the  line  of  puncture.  Upon  potato,  at  373  C  ,  a  scanty  development 
occurs  in  the  form  of  a  small,  dry,  pale-brown  mass.  Upon  blood  serum 
isolated  or  confluent,  colorless  colonies  are  formed  the  size  of  a  poppy  seed ; 
these  are  closely  adherent  lo  the  surface  of  the  culture  medium. 

Pathogenesis.- — Wben  injected  subcutaneously  into  the  ear  of  a  rabbit  it 
produces  tumefaction  and  redness;  in  guinea-pigs,  formation  of  pus.  When 
injected  into  the  circulation  of  dogs,  after  injury  to  the  aortic  valves,  an  en- 
docarditis is  developed. 

MICROCOCCUS   OF  GANGRENOUS  MASTITIS  IN   SHEEP. 

Obtained  by  Nocard  (1887)  from  the  milk  of  sheep  suffering  from  gan- 
grenous mastitis  (rnal  de  pis  or  d'araignee),  a  fatal  disease  which  attacks 
especially  sheep  which  are  being  milked  for  the  manufacture  of  cheese,  at 
Roquefort  and  elsewhere  in  France. 

Morphology. — Micrococci,  solitary,  in  pairs,  or  in  irregular  groups,  resem- 
bling the  staphylococci  of  pus  in  dimensions  and  arrangement. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing micrococcus.  Grows  at  the  room  temperature  in  the  usual  culture  me- 
dia. Upon  gelatin  plates,  at  the  end  of  forty-eight  hours,  the  colonies  are 
spherical  and  white  in  color;  under  a  low  power  the  superficial  colonies  are 
circular  in  outline,  homogeneous,  and  brown  in  color ;  they  are  surrounded 
by  a  semi-transparent  aureole  ;  liquefaction  around  the  superficial  colonies 
occurs  sooner  than  around  those  beneath  the  surface  of  the  gelatin.  In  i 
gelatin  stick  cultures,  at  18°  to  20°  C.,  on  the  second  day  liquefaction  of  the! 
gelatin  commences  near  the  surface  ;  by  the  fifth  day  a  pouch  of  liquefied ' 
gelatin  has  formed,  which  has  the  shape  of  an  inverted  cone;  at  the  bottom 
of  this  an  abundant  deposit  of  micrococci  is  seen,  while  the  liquefied  gela- 
tin above  is  clouded  throughout.  In  agar  stick  cultures  development  oc- 
curs upon  the  surface  as  a  thick  white  layer,  which  gradually  extends 
over  the  entire  surface,  and  after  a  time  acquires  a  yellowish  tint ;  develop- 
ment also  occurs  along  the  line  of  puncture.  Upon  potato  a  thin,  viscid, 
grayish  layer  is  slowly  developed ;  the  outline  is  irregular  and  the  edges 
thicker  than  the  central  portion  ;  the  central  portion  of  this  layer  gradually 


NOT   DESCRIBED   IN  SECTIONS  V.    AND  VI. 


417 


acquires  a  yellow  color,  while  the  periphery  remains  of  a  dirty-white  or 
grayish  color.     Blood  serum  is  liquefied  by  this  micrococcus. 

Pathogenesis. — A  few  drops  of  a  pure  culture  injected  subcutaneously  or 
into  the  mammary  giand  of  sheep  cause  an  extensive  inflammatory  oedema 
and  the  death  of  the  animal  in  from  twenty-four  to  forty-eight  hours.  A 
cubic  centimetre  injected  into  the  mammary  gland  of  a  goat  produced  110  re- 
sult ;  the  horse,  the  calf,  the  pig,  the  cat,  chickens,  and  guinea-pigs  also  proved 
to  be  immune.  Subcutaneous  injections  in  rabbits  produce  an  extensive  ab- 
scess at  the  point  of  inoculation. 

STREPTOCOCCUS  OF  MASTITIS  IN  COWS. 

Obtained  by  Nocard  and  Mollereau  (1887)  from  the  milk  of  cows  suffering 
from  a  form  of  chronic  mastitis  (mammite  coiitagieuse) . 

Morphology. — Spherical  or  oval  cocci,  a  little  less  than  one  //  in  diameter, 
usually  united  in  long  chains. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  streptococcus.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  Develops  rapidly  in  milk  or  in  bouillon  at  a  temperature  of 
16°  to  30"  C.  The  milk  of  a  cow  suffering  from  the  form  of  mastitis  produced 
by  this  micrococcus,  when  drawn  with  proper  precautions  in  sterilized  test 
tubes,  at  the  end  of  twenty  four  hours  is  acid  in  reaction;  the  lower  two- 
thirds  of  the  tube  is  filled  with  an  opaque,  dirty- white,  homogeneous  deposit, 
and  above  this  is  a,n  opalescent,  serous  fluid  of  a  bluish  or  dirty-yellow  or 
slightly  reddish  color,  according  to  the  age  of  the  lesion.  A  drop  of  this 
milk  examined  under  the  microscope  shows  the  presence  of  the  streptococcus 
in  great  numbers.  The  addition  of  two  to  five  per  cent  of  glucose  or  of  gly- 


Fio.  97.— Streptococcus  of  mastitis  in  cows  (Nocard). 

eerin  to  bouillon  makes  it  a  more  favorable  culture  medium ;  the  reaction 
should  be  neutral  or  slightly  alkaline,  as  this  streptococcus  does  not  grow 
readily  in  an  acid  medium,  although  it  produces  an  acid  reaction  in  media 
containing  sugar,  the  acid  formed  being  lactic.  In  gelatin  stab  cultures  the 
growth  upon  the  surface  is  scanty,  in  the  form  of  a  thin  pellicle  around  the 
point  of  puncture ;  along  the  line  of  inoculation  minute,  opaque,  granular 
colonies  are  developed,  which,  being  closely  crowded,  form  a  thick  line  with 
jagged  margins. 

In  agar  stab  cultures  the  growth  is  similar  but  more  abundant.    Upon 
the  surface  of  nutrient  gelatin,  agar,  or  blood  serum  a  large  number  of  mi- 
27 


418  PATHOGENIC  MICROCOCCI 

nute,  spherical,  semi-transparent  colonies  are  developed  among-  the  impfstrich ; 
these  have  a  bluish  tint  by  reflected  light ;  they  may  become  confluent,  form- 
ing a  thin  layer  with  well-defined  margins.  Upon  gelatin  plates,  at  16°  to 
18°  C.,  colonies  are  first  visible  at  the  end  of  two  or  three  days;  they  are 
spherical  and  slightly  granular,  at  first  transparent  and  later  of  a  pale-yellow 
color  by  transmitted  light,  which  gradually  becomes  brown.  At  the  end  of 
five  or  six  weeks  the  colonies  are  still  quite  small,  well  defined,  and  opaque. 
Pathogenesis. — Pure  cultures  injected  into  the  mammary  gland  of  cows 
and  goats  gave  rise  to  a  mastitis  resembling  in  its  development  that  from 
which  the  streptococcus  was  obtained  in  the  first  instance.  Injections  into 
the  cavity  of  the  abdomen  or  into  a  vein,  of  one  cubic  centimetre  of  a  pure 
culture,  gave  a  negative  result  in  dogs,  cats,  rabbits,  and  guinea-pigs. 

DIPLOCOCCUS  OF  PNEUMONIA  IN   HORSES. 

Obtained  by  Schiitz  (1887)  from  the  lungs  of  horses  affected  with  pneu- 
monia. 

Morphology. — Oval  cocci,  usually  in  pairs,  surrounded  by  a  homogene- 
ous, transparent  capsule. 

Does  not  stain  by  Gram's  method. 

Biological  Characters. — An  aerobic,  non-liquefying  micrococcus.  Grows 
at  the  room  temperature.  Upon  gelatin  plates  forms  small,  spherical,  white 
colonies. 

In  gelatin  stick  cultures  grows  along  the  line  of  puncture  as  small,  white, 
separate  colonies,  which  grow  larger  without  becoming  confluent.  Upon 
the  surface  of  agar  small  transparent  drops  are  developed  along  the  impf- 
strich. 

Pathogenesis. — The  injection  of  a  pure  culture  into  the  lung  of  a  horse 
produces  pneumonia  and  causes  its  death  in  eight  or  nine  days.  Pathogenic 
for  rabbits,  guinea-pigs,  and  mice. 

STREPTOCOCCUS  CORYZ^E    CONTAGIOS.E  EQUORUM. 

Obtained  by  Schiitz  (1888)  from  pus  from  the  lymphatic  glands  involved 
in  horses  suffering  from  the  disease  known  in.  Germany  as  Druse  des 
Pferdes. 

Morphology. — Oval  cocci,  in  pairs,  in  chains  containing  three  or  four 
elements,  or  in  long  chaplets. 

Stains  with  the  usual  aniline  colors — very  intensely  with  Weigert's  or 
Ehrlich's  solution. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic  micrococ- 
cus. Grows  slowly  at  the  room  temperature,  more  rapidly  at  37'  C.  Upon 
gelatin  plates  at  the  end  of  three  to  five  days  minute  colonies  become  visible ; 
these  never  exceed  the  size  of  a  pin's  head.  In  gelatin  stab  cultures  growth 
upon  the  surface  is  scanty  or  absent;  along  the  line  of  puncture  minute 
colonies  are  developed  in  rows.  Upon  agar  plates,  at  37°  C.,  at  the  end  of 
twenty  four  hours  lentil-shaped  colonies  are  developed  the  size  of  a  pin's 
head ;  under  a  low  power  the  superficial  colonies  are  seen  to  have  a  well-de- 
fined, opaque  nucleus  surrounded  by  a  grayish,  transparent  marginal  zone, 
which  represents  a  half -fluid,  slimy  growth  which  does  not  extend  after  the 
third  day  and  later  disappears  entirely;  the  deep  colonies  are  at  first  well- 
defined,  and  later  surrounded  by  wing-like  outgrowths.  Upon  blood  serum, 
at  37°  C.,  yellowish,  transparent  drops  are  first  developed;  these  become  con- 
fluent and  form  a  viscid  and  tolerably  thick  layer;  this  later  becomes  dry 
and  iridescent. 

Pathogenesis. — Pathogenic  for  horses  and  for  mice,  producing  in  these 
animals  an  abscess  at  the  point  of  inoculation,  and  metastatic  abscesses  in 
the  neighboring  lymphatic  glands.  Not  pathogenic  for  rabbits,  guinea-pigs, 
or  pigeons. 


NOT   DESCRIBED   IN   SECTIONS  V.    AND  VL  419 


HJEMATOCOCCUS  sovis  (Babes). 

Obtained  by  Babes  (1889)  from  the  blood  and  various  organs  of  cattle 
which  had  died  of  an  epidemic  malady  (in  Roumaiiia)  characterized  by  heemo- 
globinuria.  The  cocci  are  found  in  the  blood  in  great  numbers,  for  the  most 
part  enclosed  in  the  red  corpuscles. 

Morphology. — Biscuit-shaped  cocci  united  in  pairs;  sometimes  oblong  in 
form,  isolated  or  united  in  groups ;  the  free  cocci  are  surrounded  by  a  pale- 
yellowish,  shining  aureole  of  0.5  to  1  ja  in  diameter. 

Stains  best  with  Loffler's  solution  of  methylene  blue ;  does  not  stain  by 
Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  micrococcus.  Grows  very  slowly  at  the  room  temperature — not 
below  20 J  C.  In  the  incubating  oven  grows  in  the  usual  culture  media.  In 
gelatin  stab  cultures  a  scanty  development  of  small,  white  colonies  occurs 
along  the  line  of  puncture.  Upon  the  surface  of  agar  small,  transparent 
drops  are  developed  along  the  impfstrich.  Upon  potato,  at  37°  C..  a  thin, 
broad,  yellowish,  shining  layer  is  developed  in  the  course  of  a  few  days — 
scarcely  visible.  Upon  blood  serum  small,  moist,  transparent  colonies  are 
developed. 

Pathogenesis. — Pathogenic  for  rabbits  and  rats,  which  die  in  from  six  to 
ten  days  after  inoculation  with  a  pure  culture;  the  spleen  is  found  to  be  en- 
larged, the  lungs  hyperaemic,  and  a  bloody  serum  is  found  in  the  cavity  of 
the  abdomen;  the  cocci  are  present  in  the  blood  inconsiderable  numbers, 
but  are  rarely  seen  in  the  red  corpuscles.  Inoculations  in  oxen,  horses, 
goats,  sheep,  guinea-pigs,  and  birds  were  without  effect. 

STREPTOCOCCUS  PERNICIOSUS   PSITTACORUM. 

Micrococcus  of  gray  pirrot  disease.  Eberth  and  Wolff  have  described 
an  infectious  disease  of  gray  parrots,  which  is  said  to  be  extremely  fatal 
among  the  imported  birds.  The  disease  is  characterized  by  the  formation  of 
nodules  upon  the  surface  and  in  the  interior  of  various  organs,  and  especially 
in  the  liver.  Micrococci  of  medium  size  are  found  in  these  nodules  and  in 
blood  from  the  heart;  these  are  sometimes  in  chains.  Microscopic  examina- 
tion of  stained  sections  shows  that  these  cocci  are  directly  related  to  the  tis- 
sue necrosis  which  characterizes  the  disease.  But  the  micrococcus  has  not 
been  cultivated  and  its  biological  characters  are  undetermined. 

STREPTOCOCCUS  AGALACTI^  CONTAGIOS^E. 

Obtained  by  Adametz  (1894)  from  the  milk  of  cows  suffering  from  mas- 
titis (Gelben  Gait).  According  to  Adametz  all  of  the  streptococci  which 
have  been  described  by  different  investigators  (Kitt,  Nocard  and  Mollereau, 
Guillebeau,  and  others)  are  probably  varieties  of  a  single  species. 

Morphology. — Spherical  cocci  in  short  chains — \  p  in  diameter. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  streptococcus. 

Upon  gelatin  plates  forms  flat,  transparent,  white  or  bluish-white, 
slimy  colonies,  having  a  slight  pearly  lustre  and  an  irregular  outline.  In 
nutrient  gelatin  containing  five  per  cent  of  milk  sugar  the  colonies,  at  the 
end  of  eight  days,  have  a  diameter  of  0.85  to  1  millimetre;  they  are  milk- 
white  and  of  a  semi-fluid,  slimy  consistence. 

Upon  agar  plates  the  deep  colonies  are  jmnctiform  and  white  in  color- 
under  a  low  power  they  are  seen  to  have  an  irregular  dentate  contour  and  a 
brownish  color;  the  superficial  colonies  gradually  assume  the  appearance 
of  transparent,  flat  drops  having  a  diameter  of  0.5  to  0.7  millimetre.  In 
sterilized  milk  fermentation  occurs,  at  37 D  C.,  in  from  twenty  to  twenty-four 


420  PATHOGENIC  MICROCOCCI 

hours  ;  some  hours  later  the  casein  is  precipitated,  fine  gas  bubbles  are  seen 
in  the  lower  part  of  the  fluid  and  a  foam  upon  the  surface;  the  reaction  is 
acid  and  the  casein  is  not  peptonized.  The  power  of  producing  acid  and  gas 
is  diminished  or  lost  after  a  few  successive  cultures  have  been  made. 

Streptococcus  mastitis  sporadic^  (Guillebeau)  is  said  by  Adametz  to  be 
distinguished  from  the  streptococcus  above  described  (No.  44A)  by  being 
smaller — 0.5  //  in  diameter — and  by  the  fact  that  the  cultures  do  not  lose  the 
power  of  producing  fermentation  in  milk. 


MICROCOCCUS  MELITENSIS. 

Surgeon-Major  Bruce,  of  the  British  army,  in  1887  demonstrated 
the  etiological  relation  of  a  micrococcus,  now  known  as  Micrococcus 
melitensis,  to  the  infectious  disease  known  as  Malta  fever  (syn- 
onyms: Mediterranean  fever;  Neapolitan  fever;  Rock  fever  of  Gib- 
raltar, etc.).  Subsequent  researches  show  that  this  fever  is  not  re- 
stricted to  the  Mediterranean  region,  and  it  will  probably  be  found 
to  have  an  extensive  area  of  prevalence  on  both  continents.  Cases 
have  been  recognized  in  America  and  by  medical  officers  of  the  army 
stationed  in  the  Philippine  Islands.  Curry  (Captain  and  Assistant 
Surgeon  United  States  Volunteers),  in  a  recent  report  to  the  Surgeon- 
General  of  the  army,  says : 

"I  had  the  honor  to  report  to  the  Surgeon-General  of  the  Army  on  Jan- 
uary 2d,  1900,  four  cases  of  Mediterranean  or  Malta  fever,  which  came  under 
my  observation,  while  on  duty  as  pathologist  to  the  1st  Reserve  Hospital  in 
Manila,  P.  I.,  cases  occurring  among  our  troops  and  originating  on  the 
Island  of  Luzon. 

"Later,  in  a  report  on  the  '  Diseases  of  the  Philippine  Islands,'  I  reported 
twelve  additional  cases.  In  all  these  cases  a  positive  serum  reaction  with 
the  Micrococcus  melitensis  was  obtained,  and  the  clinical  history  of  the  cases 
corresponds  with  the  descriptions  of  Malta  fever  as  given  by  the  English 
army  surgeons  Bruce,  Hughes,  Wright,  Semple,  and  others,  and  that  de- 
scribed by  Manson.  Included  in  these  sixteen  cases  is  one  autopsy. 

"In  my  report  on  the  ' Diseases  of  the  Philippine  Islands,'  under  the  head- 
ing of  'Fevers  of  the  Philippines,'  I  expressed  the  belief  that  'Malta  fever 
is  not  an  uncommon  disease  in  the  Philippine  Islands,'  and  that  it  appeared 
that  'Malta  fever  is  by  no  means  as  limited  geographically  as  has  been 
thought  heretofore.' 

"Our  experience  here  in  the  Army  and  Navy  General  Hospital,  Hot 
Springs,  Ark. ,  has  convinced  me  that  Malta  fever  is  widespread  in  tropical 
and  sub-tropical  regions.  We  are  but  having  a  repetition  of  the  experience 
of  the  English  army  surgeons  at  the  Royal  Victoria  Hospital,  Netley. 

"Among  the  soldiers  and  sailors,  here  in  our  wards,  who  have  been 
returned  from  tropical  stations,  we  have  found  already  four  to  have  Malta 
fever.  These  four  cases  came  from  widely  separated  stations.  Two  cases 
are  in  soldiers,  one  from  the  Philippines,  and  one  from  Cuba,  and  two  are 
among  sailors  of  the  United  States  navy  who  were  recently  returned  from 
South  Atlantic  stations. 

"All  four  cases  entered  this  hospital  with  a  diagnosis  of  rheumatism." 

Morphology. — Micrococci,  about  0.5  i*.  in  diameter,  usually  soli- 
tary or  in  pairs ;  occasionally  short  chains  are  seen  in  cultures.  In 


NOT   DESCRIBED   IN   SECTIONS   V.    AND  VI.  421 

old  cultures  kept  at  the  room  temperature  the  cells  may  be  oval  or 
elongated. 

Biological  Characters. — An  aerobic,  non-liquefying  micro- 
coccus.  Does  not  stain  by  Gram's  method.  Grows  best  in  nutrient 
agar.  In  stab  cultures  no  growth  is  seen  for  several  days.  "At 
length  the  growth  appears  as  pearly-white  spots  scattered  around  the 
point  of  puncture  and  minute,  round,  white  colonies  are  also  seen 
along  the  course  of  the  needle  track";  these  increase  in  size,  and 
after  some  weeks  a  rosette-shaped  growth  is  seen  upon  the  surface, 
and  the  growth  along  the  line  of  puncture  has  a  yellowish-brown 
color.  At  the  end  of  nine  or  ten  days,  at  37°  C.,  some  of  the  colonies 
on  the  surface  of  nutrient  agar  are  as  large  as  No.  4  shot;  by  trans- 
mitted light  they  have  a  yellowish  color  at  the  centre,  and  the  per- 
iphery is  bluish-white;  by  reflected  light  they  have  a  milky- white 
color.  At  25°  C.  colonies  first*  become  visible  at  the  end  of  about 
seven  days,  at  37°  C.  in  three  to  four  days.  Does  not  grow  upon 
potato.  Very  scanty  growth  upon  nutrient  gelatin  at  22°  C.  at  the 
end  of  a  month. 

This  micrococcus  has  usually  been  described  as  non-motile,  but 
Gordon  has  demonstrated  that  it  has  from  one  to  four  flagella,  which 
are  difficult  to  demonstrate  by  the  usual  staining  methods. 

Pathogenesis. — Pathogenic  for  monkeys,  which  suffer  from  fever 
as  a  result  of  subcutaneous  inoculations  and  usually  die  in  from  thir- 
teen to  twenty-one  days.  The  spleen  is  found  to  be  enlarged  and 
contains  the  micrococcus.  Not  pathogenic  for  mice,  guinea-pigs, 
or  rabbits. 

In  man  the  micrococcus  is  found  in  large  numbers  in  the  spleen, 
which  is  greatly  enlarged. 

Widal  Reaction. — The  blood  serum  of  patients  suffering  from 
Malta  fever  and  of  individuals  who  have  recently  recovered  from  the 
disease  causes  the  agglutination  of  Micrococcus  melitensis  in  recent 
cultures.  According  to  Wright  and  Smith  this  reaction  may  be 
manifested  a  year  after  recovery.  Dilution  of  1 : 1000  will  in  ex- 
ceptional cases  give  a  distinct  agglutinating  effect. 


VIII. 

THE  BACILLUS  OF  ANTHRAX. 
[Fr.,  CHARBON ;    Ger.,  MILZBRAND.] 

ANTHRAX  is  a  fatal  infectious  disease  which  prevails  extensively 
among  sheep  and  cattle  in  various  parts  of  the  world,  causing  heavy 
losses.  In  Siberia  it  constitutes  a  veritable  scourge  and  is  known 
there  as  the  Siberian  plague  ;  it  also  prevails  to  a  considerable  extent 
in  portions  of  France,  Hungary,  Germany,  Persia,  and  India,  and 
local  epidemics  have  occasionally  occurred  in  England,  where  it  is 
known  under  the  name  of  splenic  fever.  It  does  not  prevail  in  the 
United  States.  In  infected  districts  the  greatest  losses  are  incurred 
during  the  summer  season. 

In  man  accidental  inoculation  may  occur  among  those  who  come 
in  contact  with  infected  animals,  and  especially  during  the  removal  of 
the  skin  and  cutting  up  of  dead  animals,  when  there  is  any  cut  or 
abrasion  upon  the  hands.  A  malignant  pustule  is  developed  as  the 
result  of  such  inoculation,  but,  as  a  rule,  general  infection  does 
not  occur,  as  is  the  case  when  inoculations  are  made  into  the  more 
susceptible  lower  animals — rabbit,  guinea-pig,  mouse.  Those  who 
handle  the  hair,  hides,  or  wool  of  infected  animals  are  also  liable  to 
contract  the  disease  by  inoculation  through  open  wounds,  or  by  the 
inhalation  of  dust  containing  spores  of  the  anthrax  bacillus.  Cases 
of  pulmonic  anthrax,  known  formerly  in  England  as  "wool-sorters' 
•disease,"  have  been  occasionally  observed  in  England  and  in  Ger- 
many, and  are  now  recognized  as  being  due  to  infection  through  the 
lungs  in  the  manner  indicated. 

The  French  physician  Davaine,  who  had  observed  the  anthrax 
bacillus  in  the  blood  of  infected  animals  in  1850,  communicated  to 
the  French  Academy  of  Sciences  the  results  of  his  inoculation  experi- 
ments in  1863  and  1864,  and  asserted  the  etiological  relation  of  the 
bacillus  to  the  disease  with  which  his  investigations  showed  it  to  be 
constantly  associated.  This  conclusion  was  vigorously  contested  by 
conservative  opponents,  but  has  been  fully  established  by  subsequent 
investigations,  which  show  that  the  bacillus,  in  pure  cultures,  induces 


THE   BACILLUS   OF   ANTHRAX.  423 

anthrax  in  susceptible  animals  as  certainly  as  does  the  blood  of  an 
animal  recently  dead  from  the  disease. 

Owing  to  the  fact  that  this  was  the  first  pathogenic  bacillus  cul- 
tivated in  artificial  media,  and  to  the  facility  with  which  it  grows  in 
various  media,  it  has  served  more  than  any  other  microorganism  for 
researches  relating  to  a  variety  of  questions  in  pathology,  general 
biology,  and  public  hygiene,  some  of  which  are  discussed  in  other 
sections  of  this  volume. 

BACILLUS   ANTHRACIS. 

Synonyms. — Milzbrandbacillus,  Ger.;  Bacteridie  du  charbon,  Fr. 

First  observed  in  the  blood  of  infected  animals  by  Pollender  (1849) 

and  by  Davaine  (1850).     Etiological  relation  affirmed  by  Davaine 


FIG.  98.— Bacillus  anthracis,  from  a  culture,  showing  development  of  long  threads  in  convo- 
luted bundles,    x  300.    (Klein.) 

(1863),  and  established  by  the  inoculation  of  pure  cultures  by  Pasteur 
(1879)  and  by  many  other  investigators. 

Morphology. — Rod-shaped  bacteria  having  a  breadth  of  1  to 
1.25  /*,  and  5  to  20  ^  in  length;  or,  in  suitable  culture  media,  growing- 
out  into  long,  flexible  filaments,  which  are  frequently  united  in 
twisted,  cord-like  bundles.  These  filaments  in  hanging-drop  cul- 
tures, before  the  development  of  spores,  appear  to  be  homogeneous  ; 
or  the  protoplasm  is  clouded  and  granular,  but  without  distinct  seg- 
mentation. But  in  stained  preparations  the  filaments  are  seen  to  be 
made  up  of  a  series  of  rectangular,  deeply  stained  segments.  In 
hanging-drop  cultures  the  ends  of  the  rods  appear  rounded,  but  in 
stained  preparations  from  the  blood  of  an  infected  animal  they  are 
seen  to  present  a  slight  concavity,  and  a  lenticular  interspace  is 
formed  where  two  rods  come  together.  The  diameter  of  the  rods 


424 


THE   BACILLUS   OF   ANTHRAX. 


varies  considerably  in  different  culture  media;  and  in  old  cultures 
irregular  forms  are  frequently  seen — "involution  forms." 

Under  favorable  conditions  endogenous  spores  are  developed  in 
the  long  filaments  which  grow  out  in  artificial  culture  media. 
These  first  appear  as  refractive  granules  distributed  at  regular  inter- 
vals in  the  segments  of  the  protoplasm,  which  gradually  disappear 
as  the  spores  are  developed ;  and  these  are  left  as  oval,  highly  re- 
fractive bodies,  held  together  in  a  linear  series  by  the  cellular  enve- 
lope, and  subsequently  set  free  by  its  dissolution.  The  germination 
of  these  reproductive  bodies  results  in  the  formation  of  rods  and 
spore-bearing  filaments  like  those  heretofore  described.  In  this  pro- 
cess the  spore  is  first  observed  to 
lose  its  brilliancy,  from  the  ab- 
sorption of  moisture,  a  promi- 
nence occurs  at  one  end  of  the 
oval  body,  and  soon  the  external 
envelope  —  exosporium — is  rup- 
tured, permitting  the  softened 
protoplasmic  contents  enclosed 
in  the  internal  spore  membrane 
— endosporium — to  escape  as  a 
short  rod,  to  which  the  empty 
exosporium  sometimes  remains 
attached. 

The  anthrax  bacillus  stains 
readily  with  the  aniline  colors 
and  also  by  Gram's  method, 
when  not  left  too  long  in  the 
decolorizing  iodine  solution. 
Loffler's  solution  of  methylene 
blue  is  an  especially  good  stain- 
ing fluid  for  this  as  well  as  for  many  other  bacilli.  Bismarck  brown 
is  well  adapted  for  specimens  which  are  to  be  photographed,  and  also 
for  permanent  preparations,  as  it  is  less  liable  to  fade  than  the  blue 
and  some  other  aniline  colors. 

Biological  Characters. — The  anthrax  bacillus  is  aerobic,  but 
not  strictly  so,  as  is  shown  by  the  fact  that  it  grows  to  the  bottom  of 
the  line  of  puncture  in  stab  cultures  in  solid  media.  It  is  non-mo- 
tile, and  is  distinguished  by  this  character  from  certain  common 
bacilli  resembling  it  in  morphology — Bacillus  subtilis — which  were 
frequently  confounded  with  it  in  the  earlier  days  of  bacteriological 
investigation. 

The  anthrax  bacillus  grows  in  a  variety  of  nutrient  media  at  a 


FIG.  99.— Bacillus  anthracis,  from  a  culture,  show- 
ing formation  of  spores.    X  1,000.    (Klein.) 


THE    BACILLUS    OF   AXTHRAX. 


425 


temperature  of  20°  to  38°  C.     Development  ceases  at  temperatures 
below  12°  C.  or  above  45°  C. 

This  bacillus  grows  best  in  neutral  or  slightly  alkaline  media,  and 
its  development  is  arrested  by  a  decidedly  acid  reaction  of  the  cul- 
ture medium.  It  may  be  cultivated  in  infusions  of  flesh  or  of  vari- 
ous vegetables,  in  diluted  urine,  in  milk,  etc. 

In  gelatin  plate  cultures  small,  white,  opaque  colonies  are  devel- 
oped in  from  twenty-four  to  thirty-six  hours,  which  under  the  micro- 
scope are  seen  to  be  somewhat  irregular  in  outline  and  of  a  greenish 
tint ;  later  the  colonies  spread  out  upon  the  surface  of  the  gelatin, 
and  the  darker  central  portion  is  surrounded  by  a  brownish  mass  of 
wavy  filaments,  which  are  associated  in  tangled  bundles.  Mycelial- 
like  outgrowths  from  the  periphery  of 
the  colony  may  often  be  seen  extending 
into  the  surrounding  gelatin.  At  the 
end  of  two  or  three  days  liquefaction  of 
the  gelatin  commences,  and  the  colony 
is  soon  surrounded  by  the  liquefied  me- 
dium, upon  the  surface  of  which  it  floats 
as  an  irregular  white  pellicle.  In  gela- 
tin stab  cultures  growth  occurs  all 
along  the  line  of  puncture  as  a  white  cen- 
tral thread,  from  which  lateral  thread- 
like ramifications  extend  into  the  culture 
medium.  At  the  end  of  two  or  three 
days  liquefaction  of  the  culture  medium 
commences  near  the  surface,  where  the 
development  has  been  most  abundant. 
At  first  a  pasty,  white  mass  is  formed, 
but  as  liquefaction  progresses  the  upper 
part  of  the  liquefied  gelatin  becomes 
transparent  from  the  subsidence  of  the 
motionless  bacilli,  and  these  are  seen 
upon  the  surface  of  the  non-liquefied 
portion  of  the  medium  in  the  form  of 
cloudy,  white  masses,  while  below  the  line  of  liquefaction  the  charac- 
teristic branching  growth  may  still  be  seen  along  the  line  of  puncture. 

In  agar  plate  cultures,  in  the  incubating  oven  at  35°  to  37°  C., 
colonies  are  developed  within  twenty-four  hours,  which  under  the 
microscope  are  seen  to  be  made  up  of  interlaced  filaments  and  are 
very  characteristic  and  beautiful.  Upon  the  surface  of  nutrient  agar 
a  grayish-white  layer  is  formed,  which  may  be  removed  in  ribbon-like 
strips  ;  and  in  stick  cultures  in  this  medium  a  branching  growth  is 
seen,  like  that  in  gelatin,  but  without  liquefaction.  The  addition  of 


FIG.  100.— Culture  of  Bacillus  an- 
thracis  in  nutrient  gelatin  :  a,  end 
of  four  days ;  6,  end  of  eight  days. 
( Baumgarten.) 


426 


THE   BACILLUS   OF   ANTHRAX. 


a  small  quantity  of  agar  to  a  gelatin  medium  prevents  liquefaction 
of  the  gelatin  (Fliigge). 

Upon  blood  serum  a  rather  thick,  white  layer  is  formed  and 
liquefaction  slowly  occurs. 

Upon  potato  the  growth  is  abundant  as  a  rather  dry,  grayish- 
white  layer,  of  limited  extent,  having  a  somewhat  rough  surface  and 
irregular  margins. 

Spores  are  formed  only  in  the  free  presence  of  oxygen,  as  in  sur- 
face cultures  upon  potato  or  nutrient  agar,  or  in  shallow  cultures  in 
liquid  media,  and  at  a  temperature  of  20°  to  35°  C.  They  are  not 
formed  during  the  development  of  the  bacilli  in  the  bodies  of  living 


FIG.  101.— Colonies  of  Bacillus  anthracis  upon  gelatin  plates  :  a,  at  end  of  twenty-four  hours; 
6,  at  end  of  forty-eight  hours,  x  80.  (Flugge.) 

animals,  but  after  the  death  of  the  animal  the  bacillus  continues  to 
multiply  for  a  time,  and  spores  may  be  formed  where  the  fluids 
containing  it  come  in  contact  with  the  air — as,  for  example,  in 
bloody  discharges  from  the  nostrils  or  from  the  bowels  of  the  dead 
animal. 

Varieties  incapable  of  spore  production  have  been  produced  arti- 
ficially, by  several  bacteriologists,  by  cultivating  the  bacillus  under 
unfavorable  conditions.  Roux  was  able  to  produce  a  sporeless  va- 
riety by  successive  cultivation  in  media  containing  a  small  quantity 
of  carbolic  acid — 1  : 1,000. 

Varieties  differing  in  their  pathogenic  power  may  also  be  pro- 
duced by  cultivation  under  unfavorable  conditions.  Thus  Pasteur 


THE  BACILLUS   OF   ANTHRAX.  427 

produced  an  "  attenuated  virus"  by  keeping  his  cultures  for  a  con- 
siderable time  before  replanting  them  upon  fresh  soil,  and  supposed 
the  effect  was  due  to  the  action  of  atmospheric  oxygen.  It  seems 
probable  that  it  was  rather  due  to  the  deleterious  action  of  its  own 
products  of  growth  present  in  the  culture  media.  It  has  been 
shown  by  Chamberlain  and  Roux  that  cultivation  in  the  presence 
of  certain  chemical  substances  added  to  the  culture  medium — e.g., 
bichromate  of  potassium  0.01  per  cent — causes  an  attenuation  of 
virulence.  The  same  result  occurs  when  cultures  are  subjected  to  a 
temperature  a  little  below  that  which  is  fatal  to  the  bacillus — 50°  C. 
for  eighteen  minutes  (Chauveau);  42.5°  C.  for  two  or  three  weeks 
(Koch).  Attenuation  of  pathogenic  virulence  is  also  effected  by  cul- 
tivation in  the  body  of  a  non-susceptible  animal,  like  the  frog  (Lu- 
barsch,  Petruschky);  or  in  the  blood  of  a  rat  (Behring);  by  exposure 
to  sunlight  (Arloing);  and  by  compressed  air  (Chauveau). 

Anthrax  spores  may  be  preserved  in  a  desiccated  condition  for 
years  without  losing  their  vitality  or  pathogenic  virulence  when  in- 
oculated into  susceptible  animals.  They  also  resist  a  comparatively 
high  temperature.  Thus  Koch  and  Wolffhiigel  found  that  dry  spores 
exposed  in  dry  air  required  a  temperature  of  140°  C. ,  maintained  for 
three  hours,  to  insure  their  destruction.  But  spores  suspended  in  a 
liquid  are  destroyed  in  four  minutes  by  the  boiling  temperature, 
100°  C.  (writer's  determination). 

The  bacilli,  in  the  absence  of  spores,  according  to  Chauveau,  are 
destroyed  in  ten  minutes  by  a  temperature  of  54°  C. 

For  the  action  of  various  antiseptic  and  germicidal  agents  upon 
this  bacillus  we  must  refer  to  the  sections  especially  devoted  to  this 
subject  (Part  Second). 

Toussaint,  by  injecting  filtered  anthrax  blood  into  animals,  obtained 
evidence  that  it  contained  some  toxic  substance  which  in  his  experi- 
ments gave  rise  to  local  inflammation  without  any  noticeable  general 
symptoms.  More  recent  investigations  show  that  a  poisonous  sub- 
stance is  formed  during  the  growth  of  the  anthrax  bacillus,  and  that 
cultures  containing  this  toxin,  from  which  the  bacilli  have  been  re- 
moved by  filtration  through  porcelain,  produce  immunity  when  in- 
jected into  susceptible  animals,  similar  to  that  resulting  from  inocu- 
lations with  an  attenuated  virus.  It  is  probable  that  the  pathogenic 
power  of  the  anthrax  bacillus  depends  largely  upon  the  presence  of 
this  toxin,  and  that  the  essential  difference  between  virulent  and 
attenuated  varieties  depends  upon  the  more  abundant  production  of 
this  toxic  substance  by  the  former.  It  has  also  been  shown  that 
virulent  cultures  produce  a  larger  quantity  of  acid  than  those  which 
have  been  attenuated  by  any  of  the  agencies  above  mentioned 
(Behring). 


TIIK.    BACILLUS    OF     \\TIM;\X. 

r<ll.ho<J<>iw»ix. — The  anthrax  hacillus  is  pat  hogenic  for  cattle, 
sheep,  horses,  rahhits,  guinea-pigs,  ami  mice.  White  rats,  dogs,  and 
frogs  are  immune,  as  is  also  the  Algerian  race  of  sheep.  The  spar- 
row is  suseepfihle  to  general  infection,  hut  chickens,  under  normal 
conditions,  aw  not.  Young  animals  a. re,  as  a  rule,  more  susceptihle 
tliaii  adults  of  the  same  species.  Man  does  not  hclong  among  the 
most,  susceptihle  animals,  hut  is  suhjecf  to  local  infection  as  a,  result 
of  accidental  inoculation— malignant  pustule — and  to  pulmonic  an- 
thrax from  hrcathing  air,  containing  spores  of  the  anthrax  hacillus, 
(I iii-ing  the  sorting  of  wool  or  hair  from  infected  animals.  In  animals 
which  have  a,  partial  immunity,  natural  or  acquired,  as  a  result,  of 
inoculations  with  attenuated  virus,  the  suhcutaneous  introduction  of 
virulent  cultures  may  give  rise  to  a  limited  local  inflammatory  pro- 
cess, with  effusion  of  hloody  serum  in  which  the  hacillus  is  found  in 
considerable  numbers ;  but  the  blood  is  not  invaded,  ami  the  animal, 
after  some  slight  symptoms  of  indisposition,  recovers.  In  susceptihle 


I  i<;.  Ktt.  -HacilluH  unthracitf  In  liver  of  mouse.     X  700.    (Fl&KKtJ.) 


animaJs  injections  henea.tli  the  skin  or  into  a  vein  tfive  risc^  to  general 
infection,  and  the  bacilli  multiply  rapidly  in  the  circulating  fluid. 
Death  occurs  in  mice  within  twenty-four  hours,  and  in  rabbits,  as  a 
rule,  in  less  than  forty-eight  hours.  The  blood  of  the  heart  and 
large  vessels  maybe  found,  in  an  autopsy  made  immediately  after 
death,  to  contain  mmparatively  few  ha.cilli  ;  hut  in  the  capillaries  of 

the  various  organs,  and  especially  in  the  great  I y  enlarged  spleen,  in 
the  liver,  the  kidneys,  and  the  lungs,  they  will  he  found  in  great 
numbers,  and  well  t;iined  sections  of  these  organs  will  give  an  as- 
tonishing picture  under  the  microscope,  which  the  student  should  not 
fail  to  see  in  preparations  made  by  himself.  The  capillaries  in  many 
places  will  he  found  stulTed  full  of  ha.cilli  ;  or  they  may  even  he  nip- 


mi:   n  ACILI.I  s  OK    \\  INK  \  \  1\".» 

lured    MS    a    result    of   the   distent  inn,    and    tin*    haeilli,    tovvl  her    \\illi 
escaped  hlood  corpuscles,  will  ho  seen  ill  the  surround  in:-,  ti      n-  In 

llio  kidneys  tho  i;-|oincmli,  especially,  appear  MS  if  iujoctod  \\illt  «-o| 
«'ivd  threads,  and  hy  rupture  lliese  may  lind  Iheir  \\  a  \  ml..  I  he  inini 
ferous  tuhulos. 

These   appearances   Mild    the  tfoiioml    symptoms    indicate-    Mi.,1    llio 
disease  produced   hy  (he  introduction  of  this  haeillu      in!<>  (lie   hndie-i  <>l 
suseeplihle  animals  is    a .  ^ouuino  H(»pl  ioa'MUM.       AM    in    olhor    I'onn-*   of 
HO])li<gM'inia;  1  he  spleen   is  ft  mi  id   l«  >  he  ;•,  i  •  .il  I  \    «  nl  n  ••.  •<!  ;    M    has  a  dark 
e«.|oi-  and    is  sofl  and    I'riahle.       \\'ilh    tin     exception    theorem      pie 
KOI  1 1    no  not;ihlo   olia  1 1  •<        all  IK  >uj;'li    I  h<     h  \  er    is  a  pi     I  <  >   he    sol  now  lull 
enlarged.       In  llio   guinea   piv;  an  exteir  i\e  i  nllanillUil<  n  \    <edi  m.i.  e\ 
lei  id  in:';  In  >m  1  he  point  of    inocilhtt  ion   l< »  I  I  n    m. .   I    d(>pendoiil    pa  i  I     «  •! 
the  hodv,  is  developed  ;   (he   snhenlanei.iis   e«  .niieel  i  \'<     ti      ne    i      mlil 
Iraled    will)    hloi  idy  serum    an<l    IIMM   u   }vla  1 1  IIOIIM   upptMi  ranee        Tin 
animal  comes  IM-.X!   hi  Hie  IIH.IISC  ill  HUHCopI  ihihl  \  .  and  culliires  \vhhh 


IK',       liwlllir  ink  Mir,. 

atlennated    t  ex  lent  t  halt  hey  will  not,  kill  a  rahhit  oj    a 

-heep  may  still  kill  ag'in  n<  a   pi#  ;  or,  if  not.  may  I-.  ill  ;i  moilHO.       PaHfenr 
haB  nhown  that    the  pathogenic  power  of  the  h;willuH  may  h«    «<    iah 
linh^d    hy  inoculations   into  n    < -ephhle    animals,  and    that  an    allenn 
at<-(|  cult  un;  whicli   will    not  kill    an  adult    guinea    pi;'   ma;,    h<     fatal   lo 
ery  yf^UJJl/  UnJJIia,!  of  thlhi  Kpeeje:^  a  nd    that    culture'-'    I  roll)    the  hlood 
of  tlliH  will  li;i         .        in'Tea-e*|    palh'/rM-uir-   vii  ulef)r  ,  . 

\''-ry  ruiuutf'  fjUantiti<       <>\    .  «  nt   ' -uh  n  •  nfalhhl  •,    lalal   to 

r/jo  ^      'i  '-f;ptihl<}  aniinalH,  hut  for   ral>hitH  and   other  |<  : 
cr-j/tihhj  BIiirnalH  th(i  <juantity  injeeier)    inlluefjee;,   t),(     r-e:-ull,  and    M 


430  THE   BACILLUS   OF  ANTHRAX. 

covery  may  occur  after  subcutaneous  or  intravenous  injection  of  a 
very  small  number  of  bacilli. 

Infection  in  cattle  and  sheep  commonly  results  from  the  ingestion 
of  spores  while  grazing  in  infected  pastures.  The  bacillus  itself,  in 
the  absence  of  spores,  is  destroyed  in  the  stomach.  While  spores  are 
not  formed  in  the  bodies  of  living  animals,  their  discharges  contain 
the  bacillus,  and  this  is  able  to  multiply  in  them  and  to  form  spores 
upon  the  surface  of  the  ground  when  temperature  conditions  are 
favorable.  It  is  probable  that  this  is  the  usual  way  in  which  pastures 
become  infected,  and  that  the  bloody  discharges  from  the  bladder 
and  bowels  of  animals  suffering  from  the  disease  furnish  a  nidus  for 
the  external  development  of  these  reproductive  elements  ;  as  also  do 
the  fluids  escaping  from  the  bodies  of  dead  animals.  And  possibly, 
under  specially  favorable  conditions,  the  bacillus  may  lead  a  sapro- 
phytic  existence  for  a  considerable  time  in  the  superficial  layers  of  the 
soil. 

Buchner  has  shown  by  experiment  that  infection  in  animals  may 
result  from  respiring  air  in  which  anthrax  spores  are  in  suspension 
in  the  form  of  dust ;  and  in  man  this  mode  of  infection  occurs  in  the 
so-called  wool-sorters'  disease. 

The  question  of  the  passage  of  the  anthrax  bacillus  from  the 
mother  to  the  foetus  in  pregnant  females  has  received  considerable 
attention.  That  this  may  occur  is  now  generally  admitted,  and  ap- 
pears to  be  established  by  the  investigations  of  Strauss  and  Chamber- 
lain, Morisani,  and  others.  That  it  does  not  always  occur  is  shown, 
however,  by  the  researches  of  other  bacteriologists,  and  especially  by 
those  of  Wolff. 

Sirena  and  Scagliosi  (1894)  report,  as  the  result  of  extended  experi  • 
ments  made  by  them,  that  anthrax  spores  may  survive  in  distilled 
water  for  twenty  months;  in  moist  or  dry  earth  for  two  years  and 
nine  months;  in  sea-water  for  one  year  and  seven  months;  in  sewage 
nearly  sixteen  months. 

Marmier  (1895)  has  made  an  extended  experimental  research  to 
determine  the  nature  of  the  specific  toxin  of  the  anthrax  bacillus. 
This  he  obtains  from  cultures,  at  a  low  temperature,  in  media  con- 
taining peptone  and  glycerin.  It  has  not  the  reactions  of  an  albu- 
minoid body  and  is  not  destroyed  by  a  temperature  of  100°  C.  In 
comparatively  large  doses  it  kills  animals  susceptible  to  anthrax,  and 
by  the  administration  of  smaller  doses  immunity  may  be  established 
in  such  animals.  This  toxin  is  contained  in  the  bacterial  cells,  and 
is  obtained  by  subjecting  these  to  the  action  of  alcohol,  or  from  the 
filtrate  when  cultures  are  made  at  a  low  temperature  in  a  medium 
containing  peptone.  It  has  not,  however,  been  obtained  in  a  pure 
form,  and  its  exact  nature  has  not  been  determined. 


IX. 
THE  BACILLUS  OF  TYPHOID  FEVER. 

NUMEROUS  researches  support  the  view  that  the  bacillus  described 
by  Eberth  in  1880  bears  an  etiological  relation  to  typhoid  fever — 
typhus  abdominalis  of  German  authors;  and  pathologists  have  ac- 
cepted this  bacillus  as  the  veritable  "germ"  of  typhoid  fever,  not- 
withstanding the  fact  that  the  final  proof  that  such  is  the  case  is  still 
wanting. 

This  final  proof  would  consist  in  the  production  in  man  or  in  one 
of  the  lower  animals  of  the  specific  morbid  phenomena  which  char- 
acterize the  disease  in  question,  by  the  introduction  of  pure  cultures 
of  the  bacillus  into  the  body  of  a  healthy  individual.  Evidently  it  is 
impracticable  to  make  the  test  upon  man,  and  thus  far  we  have  no 
satisfactory  evidence  that  any  one  of  the  lower  animals  is  subject  to 
the  disease  as  it  manifests  itself  in  man.  The  experiments  of 
Frankel  and  Simmonds  show,  however,  that  this  bacillus  is  patho- 
genic for  the  mouse  and  the  rabbit.  We  shall  refer  to  the  experi- 
ments of  these  authors  later. 

Before  the  publication  of  Eberth's  first  paper  Koch  had  observed 
this  bacillus  in  sections  made  from  the  spleen  and  liver  of  typhoid 
cases,  and  had  made  photomicrographs  from  these  sections.  His 
name  is,  therefore,  frequently  associated  with  that  of  Eberth  as  one 
of  the  discoverers  of  the  typhoid  bacillus.  Other  investigators  had  no 
doubt  previously  observed  the  same  organism,  but  some  of  them  had 
improperly  described  it  as  a  micrococcus.  Such  a  mistake  is  easily 
made  when  the  examination  is  made  with  a  low  power  ;  even  with  a 
moderately  high  power  the  closely  crowded  colonies  look  like  masses 
of  micrococci,  and  it  is  only  by  focussing  carefully  upon  the  scattered 
organisms  on  the  outer  margin  of  a  colony  that  the  oval  or  rod-like 
form  can  be  recognized. 

Several  observers  had  noted  the  presence  of  microorganisms  in 
the  lesions  of  typhoid  fever  prior  to  the  publication  of  Eberth's  pa- 
per, and  Browicz  in  1875,  and  Fischel  in  1878,  had  recognized  the 
presence  of  oval  organisms  in  the  spleen  which  were  probably  identi- 
cal with  the  bacillus  of  Eberth. 

The  researches  of  Gaffky  (1884)  strongly  support  the  view  that 


432  THE   BACILLUS   OF   TYPHOID   FEVER. 

the  bacillus  under  consideration  bears  a  causal  relation  to  typhoid 
fever.  Eberth  was  only  successful  in  finding  the  bacillus  in  the 
lymphatic  glands  or  in  the  spleen  in  eighteen  cases  out  of  forty  in 
which  he  searched  for  it.  On  the  other  hand,  he  failed  to  find  it  in 
eleven  cases  of  various  nature — partly  infectious  processes — and  in 
thirteen  cases  of  tuberculosis  in  which  the  lymphatic  glands  were 
involved,  and  in  several  of  which  there  was  ulceration  of  the  mucous 
membrane  of  the  intestine. 

Koch,  independently  of  Eberth  and  before  the  publication  of  his 
first  paper,  had  found  the  same  bacillus  in  about  half  of  the  cases 
examined  by  him,  and  had  pointed  out  the  fact  that  they  were  lo- 
cated in  the  deeper  parts  of  the  intestinal  mucous  membrane,  beyond 
the  limits  of  necrotic  changes,  and  also  in  the  spleen,  whereas  the 
long,  slender  bacillus  of  Klebs  was  found  only  in  the  necrosed  por- 
tions of  the  intestinal  mucous  membrane. 

The  researches  of  W.  Meyer  (1881)  gave  a  larger  proportion  of 
successful  results.  This  author  confined  his  attention  chiefly  to  the 
swollen  plaques  of  Peyer  and  follicles  of  the  intestine  which  had  not 
yet  undergone  ulceration.  The  short  bacillus  which  had  been  de- 
scribed by  Eberth  and  Koch  was  found  in  sixteen  out  of  twenty  cases 
examined.  The  observations  of  this  author  are  in  accord  with  those 
of  Eberth  as  to  the  presence  of  the  bacillus  in  greater  abundance  in 
cases  of  typhoid  which  had  proved  fatal  at  an  early  date. 

The  fact  that  in  these  earlier  researches  the  bacilli  were  not  found 
in  a  considerable  proportion  of  the  cases  examined  is  by  no  means 
fatal  to  the  view  that  they  bear  an  etiological  relation  to  the  disease. 
As  Gaffky  says  in  his  paper  referred  to  : 

"  This  circumstance  admits  of  two  explanations.  Either  in  those 
cases  in  which  the  bacillus  has  been  sought  with  negative  results 
they  may  have  perished  collectively,  before  the  disease  process  which 
thev  had  induced  had  run  its  course  ;  or  the  proof  of  the  presence  of 
bacilli  was  wanting  only  on  account  of  the  technical  difficulties  which 
attend  the  finding  of  isolated  colonies." 

Gaffky's  own  researches  indicate  that  the  latter  explanation  is  the 
correct  one. 

In  twenty-eight  cases  examined  by  this  author  characteristic 
colonies  of  the  bacillus  were  found  in  all  but  two.  In  one  of  these, 
one  hundred  and  forty-six  sections  from  the  spleen,  liver,  and  kid- 
neys were  examined  without  finding  a  single  colony,  and  in  the  other 
a  like  result  attended  the  examination  of  sixty-two  sections  from  the 
spleen  and  twenty-one  sections  from  the  liver.  In  the  first  of  these 
cases,  however,  numerous  colonies  were  found  in  recent  ulcers  of  the 
intestinal  mucous  membrane,  deeply  located  in  that  portion  of  the 
tissue  which  was  still  intact.  These  recent  ulcers  were  in  the  neigh- 


THE   BACILLUS   OF   TYPHOID   FEVER.  433 

borhood  of  old  ulcers  and  are  supposed  to  have  indicated  a  relapse 
of  the  specific  process.  In  the  second  case  the  negative  result  is 
thought  by  Gaffky  to  have  been  not  at  all  surprising,  as  the  patient 
died  at  the  end  of  the  fourth  week  of  sickness,  not  directly  from  the 
typhoid  process,  but  as  a  result  of  perforation  of  the  intestine. 

Gaffky  has  further  shown  that  in  those  cases  in  which  colonies 
are  not  found  in  the  spleen,  or  in  which  they  are  extremely  rare,  the 
presence  of  the  bacillus  may  be  demonstrated  by  cultivation ;  and 
that,  when  proper  precautions  are  taken,  pure  cultures  of  the  bacil- 
lus may  always  be  obtained  from  the  spleen  of  a  typhoid  case. 
Hein  has  been  able  to  demonstrate  the  presence  of  the  bacillus  and 
to  start  pure  cultures  from  material  drawn  from  the  spleen  of  a  living 
patient  by  means  of  a  hypodermatic  syringe.  Philopowicz  has  re- 
ported his  success  in  obtaining  cultures  of  the  bacillus  by  the  same 
method. 

The  fact  that  a  failure  to  demonstrate  the  presence  of  microor- 
ganisms by  a  microscopic  examination  cannot  be  taken  as  proof  of 
their  absence  from  an  organ,  is  well  illustrated  by  a  case  (No.  18)  in 
which  the  bacillus  was  obtained  by  Gaffky  from  the  spleen  and  also 
from  the  liver,  in  pure  cultures  ;  whereas  in  cover-glass  preparations 
made  from  the  same  spleen  he  failed  to  find  a  single  rod,  and  more 
than  one  hundred  sections  of  the  spleen  were  examined  before  he 
found  a  colony. 

To  obtain  pure  cultures  from  the  spleen  Gaffky  first  carefully 
washes  the  organ  with  a  solution  of  mercuric  chloride,  1 : 1,000.  A 
long  incision  is  then  made  through  the  capsule  with  a  knife  sterilized 
by  heat.  A  second  incision  is  made  in  this  with  a  second  sterilized 
knife,  and  a  third  knife  is  used  to  make  a  still  deeper  incision  in  the 
same  track.  By  this  means  the  danger  of  conveying  organisms  from 
the  surface  to  the  interior  of  the  organ  is  avoided.  From  the  bottom 
of  this  incision  a  little  of  the  soft  splenic  tissue  is  taken  up  on  a  ster- 
ilized platinum  needle,  and  this  is  plunged  into  the  solid  culture 
medium,  or  drawn  along  the  surface  of  the  same,  or  added  to  lique- 
fied gelatin  and  poured  upon  a  glass  plate.  The  colonies  develop,  in 
an  incubating  oven,  in  the  course  of  twenty-four  to  forty-eight  hours. 

Gaffky  has  also  shown  that  the  bacillus  is  present  in  the  liver,  in 
the  mesenteric  glands,  and,  in  a  certain  proportion  of  cases  at  least, 
in  the  kidneys,  in  which  it  was  found  in  three  cases  out  of  seven. 

The  appearance  of  the  colonies  in  stained  sections  of  the  spleen 
is  shown  in  Figs.  104  and  105.  Two  colonies  are  seen  in  Fig.  104 
(at  a,  a)  as  they  appear  under  a  low  power — about  sixty  diameters. 
In  Fig.  105  one  of  the  colonies  is  seen  more  highly  magnified — about 
five  hundred  diameters. 

Frankel  and  Simmonds  have  demonstrated  that  the  bacilli  multi- 

28 


434 


THE   BACILLUS   OF   TYPHOID   FEVER. 


ply  in  the  spleen  after  death,  and  that  numerous  colonies  may  be 
found  in  portions  of  the  organ  which  have  been  kept  for  twenty- 
four  to  forty-eight  hours  before  they  were  placed  in  alcohol,  when 
other  pieces  from  the  same  spleen  placed  in  alcohol  soon  after  the 
death  of  the  patient  show  but  few  colonies  or  none  at  all. 

This  observation  does  not  in  any  way  weaken  the  evidence  as  to 
the  etiological  role  of  the  bacillus,  but  simply  shows  that  dead  ani- 
mal matter  is  a  suitable  nidus  for  the  typhoid  germ — a  fact  which 
has  been  repeatedly  demonstrated  by  epidemiologists  and  insisted 
upon  by  sanitarians. 

The  authors  last  referred  to  confirm  Gaffky  as  regards  the  con- 
stant presence  of  the  bacillus  in  the  spleen.  In  twenty-nine  cases 
they  obtained  it  by  plate  cultures  twenty-five  times,  and  remark 
that  in  the  four  cases  attended  with  a  negative  result  this  result  is 


FIG.  104. 


FIG.  105. 


not  at  all  surprising,  inasmuch  as  the  typhoid  process  had  termi- 
nated and  death  resulted  from  complications. 

Gaffky  did  not  succeed  in  obtaining  cultures  from  the  blood  of 
typhoid-fever  patients,  and  concludes  from  his  researches  that  if  the 
bacilli  are  present  in  the  circulating  fluid  it  must  be  in  very  small 
numbers.  He  remarks  that  possibly  the  result  would  be  different  if 
the  blood  were  drawn  directly  from  a  vein  instead  of  from  the  capil- 
laries of  the  skin.  Friinkel  and  Simmonds  also  report  that  gelatin, 
to  which  blood  drawn  from  the  forefinger  of  typical  cases  had  been 
added,  remained  sterile  when  poured  upon  plates  in  the  usual  man- 
ner— Koch's  method.  The  blood  was  obtained  from  six  different  in- 
dividuals, all  in  an  early  stage  of  the  disease — the  second  to  the 
third  week.  A  similar  experiment  made  with  blood  obtained,  post 
mortem,  from  the  large  veins  or  from  the  heart,  also  gave  a  negative 
result  in  every  instance  save  one.  In  the  exceptional  case  a  single 


THE   BACILLUS    OF    TYPHOID    FEVER.  435 

colony  developed  upon  the  plate.  In  view  of  these  results  we  are 
inclined  to  attribute  the  successful  attempts  reported  by  some  of  the 
earlier  experimenters  (Letzerich,  Almquist,  Maragliano)  to  accidental 
contamination  and  imperfect  methods  of  research.  The  more  recent 
work  of  Tayon  does  not  inspire  any  greater  confidence.  This  author 
obtained  cultures  in  bouillon  by  inoculating  it  with  blood  drawn 
from  a  typhoid  patient,  and  found  that  these  were  fatal,  in  a  few 
hours,  to  guinea-pigs,  when  injected  into  the  peritoneal  cavity.  The 
lesions  observed  are  said  to  have  resembled  those  of  typhoid  fever — 
congestion  and  tumefaction  of  Peyer's  plaques  and  of  the  mesenteric 
glands,  congestion  of  the  liver,  the  kidneys,  etc. 

The  presence  of  the  bacillus  of  Eberth  in  the  alvine  evacuations  of 
typhoid  patients  has  been  demonstrated  by  Pfeiffer  and  by  Frankel 
and  Simmonds.  This  demonstration  is  evidently  not  an  easy  mat- 
ter, for  while  the  bacilli  are  probably  always  present  in  some  portion 
of  the  intestine  during  the  progress  of  the  disease,  it  does  not  follow 
that  they  are  present  in  every  portion  of  the  intestinal  contents.  As 
only  a  very  small  amount  of  material  is  used  in  making  plate  cul- 
tures, and  as  there  are  at  all  times  a  multitude  of  bacteria  of  various 
species  in  the  smallest  portion  of  faecal  matter,  it  is  not  to  be  ex- 
pected that  the  typhoid  bacillus  will  be  found  upon  every  plate. 
Frankel  and  Simmonds  made  eleven  attempts  to  obtain  the  bacillus 
by  the  plate  method,  using  three  plates  each  time,  as  is  customary 
with  those  who  adhere  strictly  to  the  directions  of  the  master,  and 
were  successful  in  obtaining  the  bacillus  in  three  instances — in  two 
in  great  numbers  and  in  the  third  in  a  very  limited  number  of  colo- 
nies. 

The  numerous  attempts  which  have  been  made  to  communicate 
typhoid  fever  to  the  lower  animals  have  given  a  negative  result  in 
every  instance.  Murchison,  in  1867,  fed  typhoid-fever  discharges  to 
swine,  and  Klein  has  made  numerous  experiments  of  the  same  kind 
upon  apes,  dogs,  cats,  guinea-pigs,  rabbits,  and  white  mice,  without 
result.  Birch-Hirschfeld,  in  1874,  by  feeding  large  quantities  of 
typhoid  stools  to  rabbits,  produced  in  some  of  them  symptoms  which 
in  some  respects  resembled  those  of  typhoid  ;  but  these  experiments 
were  repeated  by  Bahrdt  upon  ten  rabbits  with  an  entirely  negative 
result.  Von  Motschukoffsky  met  with  no  better  success  in  his  at- 
tempts to  induce  the  disease  by  injecting  blood  from  typhoid  patients 
into  apes,  rabbits,  dogs,  and  cats.  Walder  also  experimented  with 
fresh  and  with  putrid  discharges  from  typhoid  patients,  and  with 
blood  taken  from  the  body  after  death,  feeding  this  material  to 
calves,  dogs,  cats,  rabbits,  and  fowls,  without  obtaining  any  posi- 
tive results.  Klebs  has  also  made  numerous  experiments  of  a  simi- 
lar nature,  and  in  a  single  instance  found  in  a  rabbit,  which  died 


430  THE   BACILLUS   OF   TYPHOID   FEVER. 

forty-seven  hours  after  receiving  a  subcutaneous  injection  of  a  cul- 
ture fluid  containing  his  "  typhoid  bacillus,"  pathological  lesions  re- 
sembling those  of  typhoid. 

Eberth  and  Gaffky  very  properly  decline  to  attach  any  import- 
ance to  this  solitary  case,  in  which,  as  the  first-named  writer  re- 
marks, a  different  explanation  is  possible,  and  the  possibility  of  an 
intestinal  mycosis  not  typhoid  in  its  nature  must  be  considered. 

Gaffky  has  also  made  numerous  attempts  to  induce  typhoid 
symptoms  in  animals  by  means  of  pure  cultures  of  Eberth's  bacillus, 
given  with  their  food  or  injected  into  the  peritoneal  cavity  or  subcu- 
taneously.  The  first  experiments  were  made  upon  five  Java  apes. 
For  a  considerable  time  these  animals  were  fed  daily  with  pure  cul- 
tures containing  spores.  The  temperature  of  the  animals  was  taken 
twice  daily.  The  result  was  entirely  negative.  No  better  success 
attended  the  experiments  upon  rabbits  (16),  guinea-pigs  (13),  white 
rats  (7),  house  mice  (11),  field  mice  (4),  pigeons  (2),  one  hen  and  a  calf. 

Cornil  and  Babes  report  a  similar  negative  result  from  pure  cul- 
tures of  the  typhoid  bacillus  injected  into  the  peritoneal  cavity  and 
into  the  duodenum  in  rabbits  and  guinea-pigs. 

Frankel  and  Simmonds  have  made  an  extended  series  of  experi- 
ments upon  guinea-pigs,  rabbits,  and  mice,  and  have  shown  that 
pure  cultures  of  the  bacillus  of  Eberth  injected  into  the  last-men- 
tioned animals — mice  and  rabbits — may  induce  death,  and  that  the 
bacillus  may  again  be  obtained  in  pure  cultures  from  their  organs. 
It  is  not  claimed  that  the  animals  suffer  an  attack  of  typhoid  fever 
as  the  result  of  these  injections,  but  that  their  death  is  due  to  the 
introduction  into  their  bodies  of  the  typhoid  bacillus,  and  that  this 
bacillus  is  thereby  proved  to  be  pathogenic. 

BACILLUS   TYPHI   ABDOMINALIS. 

Synonyms. — Bacillus  typhosus  ;  Typhus  bacillus. 

Eberth  (1880  and  1881)  demonstrated  the  presence  of  this  bacillus 
in  the  spleen  and  diseased  glands  of  the  intestine  in  typhoid  cada- 
vers. Gaffky  (1884)  first  obtained  it  in  pure  cultures  from  the  same 
source  and  determined  its  principal  biological  characters. 

It  is  found,  in  the  form  of  small,  scattered  colonies,  in  the  spleen, 
the  liver,  the  glands  of  the  mesentery,  the  diseased  intestinal  glands, 
and  in  smaller  numbers  in  the  kidneys,  in  fatal  cases  of  typhoid  fever; 
it  has  also  been  obtained,  by  puncture,  from  the  spleen  during  life, 
from  the  alvine  discharges  of  the  sick,  and  rarely  from  the  urine. 
It  is  not  found  in  the  blood  of  the  general  circulation,  unless,  pos- 
sibly, in  rare  cases  and  in  small  numbers. 

Morphology. — Bacilli,  usually  one  to  three  //in  length  and  about 


THE   BACILLUS   OF   TYPHOID    FEVER. 


437 


0.5  to  0.8  [j.  broad,  with  rounded  ends;  may  also  grow  out  into  long 
threads,  especially  upon  the  surface  of  cooked  potato.  The  dimen- 
sions of  the  rods  differ  considerably  in  different  media.  Spherical  or 
oval  refractive  granules  are  often  seen  at  the  extremities  of  the  rods, 
especially  in  potato  cultures  kept  in  the  incubating  oven  ;  these  are 
not  reproductive  spores,  as  was  at  first  supposed.  The  bacilli  have 
numerous  flagella  arranged  around  the  periphery  of  the  cells — usually 
from  five  to  twenty,  but  many  short  rods  have  but  a  single 


FIG.  106.  FIG.  107. 

FIG.  106.— Bacillus  typhi  abdominalis,  from  single  gelatin  colony.  X  1,000.  From  a  photo- 
micrograph.  (Frankel  and  Pfeiffer.  > 

FIG.  107.— Bacillus  typhi  abdominalis,  from  single  gelafn  colony.  X  1,000.  From  a  photo- 
micrograph. (Sternberg.) 

terminal  flagellum.  These  flagella  are  spiral  in  form,  about  0. 1  /*  in 
thickness,  and  from  three  to  five  times  as  long  as  the  rods  (Babes). 

In  stained  preparations  unstained  "  vacuoles"  may  often  be  seen 
at  the  margins  of  the  rods,  either  along  the  sides  or  at  the  ends  ; 
these  appear  to  be  due  to  a  retraction  of  the  protoplasm  from  the  cell 
membrane. 

The  typhoid  bacillus  stains  with  the  aniline  colors,  but  more 
slowly  than  many  other  bacteria,  and  easily  parts  with  its  color  when 
treated  with  decolorizing  agents — e.g.,  iodine  solution  as  employed  in 
Gram's  method.  Loffler's  solution  of  methylene  blue  is  an  excellent 
staining  agent  for  this  bacillus,  but  permanent  preparations  fade  out 
after  a  time  ;  f  uchsin,  gentian  violet,  or  Bismarck  brown,  in  aqueous 
solution,  may  also  be  used.  The  flagella  may  be  demonstrated  by 
Loffler's  method  of  staining  (p.  32). 

To  stain  the  bacillus  in  sections  of  the  spleen,  etc. ,  it  is  best  to 
leave  these  in  Loffler's  methylene  blue  solution  or  in  the  carbol- 
fuchsin  solution  of  Ziehl  for  twelve  hours  or  more ;  or  the  aniline- 


438 


THE   BACILLUS   OF   TYPHOID   FEVER. 


fuchsin  solution  may  be  used.  The  sections  should  be  washed  in 
distilled  water  only,  when  Ziehl's  solution  is  used,  or  with  a  very  di- 
lute solution  of  acetic  acid  when  Ehiiich's  tubercle  stain  is  employed 
(Baumgarten). 


FIG.  108.— Bacillus  typhi  abdominal! s,  stained  by  Loffler's  method,  showing  flagella.  x  1,000. 
From  a  photomicrograph  by  Frankel  and  Pfeiffer. 

Biological  Characters. — The  typhoid  bacillus  is  a  motile,  aero- 
bic, non-liquefying  bacillus,  which  grows  readily  in  a  variety  of 
culture  media  at  the  "  room  temperature/'  Although  it  grows  most 
abundantly  in  the  presence  of  free  oxygen,  it  may  also  develop  in  its 
absence,  and  is  consequently  a  facultative  anaerobic. 

In  gelatin  plate  cultures  small,  white  colonies  are  developed  at 
the  end  of  thirty-six  to  forty-eight  hours,  which  under  the  microscope 

are  seen  to  be  somewhat  irregular  in 
outline  and  of  a  spherical,  oval,  or  long- 
oval  form ;  these  have  by  transmitted 
light  a  slightly  granular  appearance  and 
a  yellowish-brown  color.  At  the  end  of 
three  or  four  days  the  colonies  upon  the 
surface  of  the  gelatin  form  a  grayish- 
white  layer  of  one  to  two  millimetres  in 
diameter,  with  more  or  less  irregular 
margins,  and,  when  developed  from  deep 
colonies,  with  an  opaque  central  nucleus. 

FIG.  109.— Single  colony  of  Bacillus        rrn  i       •          ~u  -j.i    j     T    T-J. 

typhi  abdominalis.in  nutrient  gela-        These    Colonies,    by    transmitted     light, 

tin.   (x?)  From  a  photograph  by      have  a  yellowish-brown  color  towards 

the    centre,   where  they  are   thickest, 
while  the  margins  are  colorless  and  transparent ;  the  surface  is  com- 


THE    BACILLUS   OF   TYPHOID    FEVER. 


439 


feu 


monly  marked  with  a  network  of  lines  and  furrows.  Stab  cultures  in 
ten-per-cent  gelatin,  at  18°  to  20°  C.,  at  the  end  of  three  days  show 
upon  the  surface  a  whitish,  semi-transparent  layer,  with  sharply 
defined  margins  and  irregular  outline,  which  has  a  shining,  pearly 
lustre ;  and  along  the  line  of  puncture  a  gray- 
ish-white growth,  made  up  of  crowded  colo- 
nies, which  are  larger  and  more  distinct  at  the 
bottom  of  the  line  of  growth.  Upon  nutrient 
cigar,  at  a  temperature  of  35°  to  37°  C.,  the 
growth  is  more  rapid  and  forms  a  whitish, 
semi-transparent  layer.  The  cultures  give  off 
a  faint  putrefactive  odor.  The  growth  upon 
blood  serum  is  rather  scanty,  in  the  form  of 
transparent,  shining  patches  along  the  line 
of  inoculation. 

The  typhoid  bacillus  develops  abundantly 
in  milky  in  which  fluid  it  produces  an  acid 
reaction ;  it  also  grows  in  various  vegetable  in- 
fusions and  in  bouillon. 

None  of  the  above  characters  of  growth  are 
distinctive,  as  certain  common  bacilli  found 
in  normal  fasces  present  a  very  similar  appear- 
ance when  cultivated  in  the  same  media. 

The  growth  of  this  bacillus  upon  potato  is 
an  important  character,  as  was  first  pointed 
out  by  Gaffky.  In  the  incubating  oven  at  the 
end  of  forty-eight  hours,  or  at  the  room  tem- 
perature in  three  or  four  days,  the  surface  of 
the  potato  has  a  moist,  shining  appearance, 
but  there  is  no  visible  growth  such  as  is  produced  by  many  other  bac- 
teria upon  this  medium.  A  simple  inspection  would  lead  to  the  belief 
that  no  growth  had  occurred ;  but  if  with  a  platinum  needle  a  little  ma- 
terial is  scraped  from  any  portion  of  the  shining  surface  and  a  stained 
preparation  is  made  from  it,  numerous  bacilli  will  be  seen,  some  of 
which  are  likely  to  be  in  the  form  of  quite  long  threads,  while  others 
are  short  and  have  rounded  extremities.  This  "  invisible  growth " 
has  been  shown  by  the  researches  of  Buchner  and  others  to  be  most 
characteristic  upon  potatoes  having  a  decidedly  acid  reaction,  as  is 
usually  the  case.  When  cultivated  upon  potatoes  having  an  alkaline 
reaction  a  thin,  visible  film  of  a  yellowish-brown  color  and  of  limited 
extent  may  be  developed.  Inasmuch  as  several  common  and  widely 
distributed  bacteria  closely  resemble  the  typhoid  bacillus  in  form  and 
in  their  growth  in  nutrient  gelatin,  this  character  of  invisible  growth 


FIG.  110.— Bacillus  typhi 
abdominalis ;  stick  culture 
in  nutrient  gelatin,  eighth 
day  at  16°-20°  C.  (Baum- 
garten.) 


440  THE   BACILLUS   OF   TYPHOID   FEVER. 

upon  potato  is  very  important  for  its  differentiation,  especially  as  the 
common  bacilli  referred  to — Bacillus  coli  communis,  bacillus  of  Em- 
merich— produce  a  very  distinct  and  rather  thick,  yellowish- white 
mass  upon  the  surface  of  potato.  But  recent  researches  show  that 
this  invisible  growth,  although  not  a  common  character,  does  not 
"belong  exclusively  to  the  typhoid  bacillus  (Babes). 

This  bacillus  in  its  development  in  culture  media  produces  acids — • 
according  to  Brieger  small  quantities  of  volatile  fat  acids,  and,  in 
presence  of  grape  sugar,  lactic  acid.  It  also  grows  readily  in  a  de- 
cidedly acid  medium,  and  this  character  has  been  employed  as  a  test 
for  differentiating  it  from  other  similar  bacilli ;  but  some  of  these 
also  grow  in  a  decidedly  acid  medium,  and  too  much  reliance  cannot 
be  placed  upon  this  test. 

Brieger  has  shown  that  indol  is  not  produced  in  cultures  of  the 
typhoid  bacillus,  and  Kitasato  has  proposed  to  use  the  indol  test  for 
differentiating  this  from  other  similar  bacilli  which  are  said,  as  a 
rule,  to  give  the  indol  reaction.  This  test  consists  in  the  addition  to 
ten  cubic  centimetres  of  a  bouillon  culture  which  has  been  in  the  in- 
cubating oven  for  twenty-four  hours,  of  one  cubic  centimetre  of  a 
solution  of  sodium  nitrite  (0.02  gramme  to  one  hundred  cubic  centi- 
metres of  distilled  water),  together  with  a  few  drops  of  concentrated 
sulphuric  acid.  If  indol  is  present  a  red  color  is  developed. 

None  of  the  above-mentioned  tests  are  entirely  reliable,  but,  taken 
together  with  the  morphological  and  biological  characters  above  de- 
scribed, they  may  enable  the  bacteriological  expert  to  give  a  tolerably 
confident  opinion  as  to  the  presence  of  this  bacillus  in  a  water  supply 
suspected  of  contamination,  etc.  And  when  a  bacillus  having  these 
characters  is  obtained  in  a  pure  culture  from  the  spleen  of  a  typhoid 
cadaver  the  student  may  be  very  sure  that  he  has  the  typhoid  bacillus. 
But  in  the  presence  of  various  similar  bacilli,  as  in  faeces,  very  careful 
comparative  researches  will  be  required  to  determine  in  a  definite 
manner  that  a  non-liquefying  bacillus  obtained  in  pure  cultures  by 
the  plate  method  is  really  the  one  now  under  consideration — espe- 
cially so  as  the  cultures  of  the  typhoid  bacillus  in  the  same  medium 
may  differ  considerably  at  different  times,  and  a  number  of  bacilli 
are  known  which  resemble  it  so  closely  that  it  is  still  uncertain 
whether  they  are  to  be  considered  as  varieties  of  the  typhoid  bacillus 
or  as  distinct  species.  Thus  Babes,  in  an  extended  research,  found  in 
the  organs  of  typhoid  cases,  associated  with  the  true  typhoid  bacillus, 
other  bacilli  or  varieties  very  closely  resembling  it.  He  has  also 
described  three  varieties  (?),  obtained  by  him  from  other  sources, 
which  could  only  be  differentiated  from  the  true  typhoid  bacillus  by 
very  careful  comparison  of  cultures  made  side  by  side  in  various 
media. 


THE    BACILLUS    OF    TYPHOID    FEVER.  441 

Cassedebat,  also,  in  an  extended  examination  of  the  river  water 
at  Marseilles  with  reference  to  the  presence  of  the  typhoid  bacillus, 
found  three  species  which  very  closely  resembled  it,  but  which  by 
careful  comparison  were  shown  to  present  slight  but  constant  dif- 
ferences in  their  biological  characters.  He  was  not  able  to  find  the 
true  typhoid  bacillus,  and  his  researches,  together  with  those  of  Babes 
and  other  recent  investigators,  make  it  appear  probable  that  numerous 
mistakes  have  been  made  by  bacteriologists  who  have  reported  the 
finding  of  the  typhoid  bacillus  in  river  and  well  water,  in  fa3ces,  etc., 
and  who  have  depended  mainly  upon  the  character  of  invisible 
growth  upon  potato  in  making  their  diagnosis.  Cassedebat  states 
that  all  three  of  his  pseudo-typhoid  bacilli  corresponded  in  their 
growth  upon  potato  with  the  bacillus  of  Eberth.  They  also  corre- 
sponded in  their  growth  on  gelatin,  agar-agar,  and  blood  serum, 
which,  as  heretofore  remarked,  has  no  characteristic  features.  They 
all  gave  a  negative  indol  reaction.  Like  the  typhoid  bacillus,  they 
grew  in  milk  without  causing  coagulation  of  the  casein,  but  two  of 
them  produced  an  alkaline  reaction  in  this  fluid,  while  the  third  cor- 
responded with  the  typhoid  bacillus  in  producing  a  decided  acid  re- 
action. Differences  were  also  observed  in  bouillon  cultures,  and  in 
bouillon  and  milk  to  which  various  aniline  colors  had  been  added,  as 
recommended  by  Holz. 

Whether  the  typhoid  bacillus,  as  obtained  from  the  spleen  of  a 
typhoid  cadaver,  is  in  truth  specifically  distinct  from  these  similar 
bacilli,  or  whether  they  are  all  varieties  of  the  same  species,  result- 
ing from  modifications  in  their  biological  characters  acquired  during 
their  continuous  development  under  different  conditions,  is  an  un- 
settled question.  But,  in  view  of  the  experimental  evidence  now 
available,  there  is  nothing  improbable  in  the  supposition  that  they  are 
simply  varieties,  and  that,  as  the  result  of  a  saprophytic  mode  of 
life,  this  bacillus  may  undergo  more  or  less  permanent  modifications. 

In  the  writer's  experiments  (1887)  the  thermal  death-point  of  the 
typhoid  bacillus  was  found  to  be  56°  C.,  the  time  of  exposure  being 
ten  minutes  ;  and  potato  cultures  containing  the  refractive  granules 
described  by  Gaffky  as  spores  were  found  to  be  infallibly  destroyed 
by  a  temperature  of  60°  C.  This  result  has  been  confirmed  by  Buch- 
ner  (1888)  and  by  Janowsky  (1890),  and  the  inference  seems  justified 
that  these  granules  are  not  reproductive  bodies,  as  was  at  first  be- 
lieved ;  for  spores  are  distinguished  by  their  great  resistance  to  heat 
and  other  destructive  agencies.  According  to  Buchner,  the  bacilli 
containing  these  refractive  granules  are  even  less  resistant  than  fresh 
cultures  in  which  they  are  not  present,  and  he  is  disposed  to  look 
upon  them  as  representing  a  degeneration  of  the  protoplasm  of  the 
cells.  They  do  not  stain  by  the  methods  which  are  successful  in 


442  THE   BACILLUS   OF   TYPHOID   FEVER. 

staining  the  spores  of  other  bacilli,  and,  in  short,  present  none  of  the 
characters  which  distinguish  spores,  except  the  form  and  high  re- 
fractive power. 

The  typhoid  bacillus  retains  its  vitality  for  many  months  in  cul- 
tures; the  writer  has  preserved  bouillon  cultures  for  more  than  a  year 
in  hermetically  sealed  tubes,  and  has  found  that  development 
promptly  occurred  in  nutrient  gelatin  inoculated  from  these.  Dried 
upon  a  cover  glass,  it  may  grow  in  a  suitable  medium  after  having 
been  preserved  for  eight  to  ten  weeks  (Pfuht).  When  added  to 
sterilized  distilled  water  it  may  retain  its  vitality  for  more  than  four 
weeks  (Bolton),  (forty  days  Cassedebat),  and  in  sterilized  sea- water 
for  ten  days  (De  Giaxa).  Added  to  putrefying  faeces  it  may  preserve 
its  vitality  for  several  months  (Ufflemann),  in  typhoid  stools  for  three 
months  (Karlinski),  and  in  earth  upon  which  bouillon  cultures  had 
been  poured  for  five  and  one-half  months  (Grancher  and  Deschamps). 

In  hanging-drop  cultures  this  bacillus  may  be  seen  to  exhibit  very 
active  movements,  the  shorter  rods  rapidly  crossing  the  field  with  a 
darting  or  to-and-fro,  progressive  motion,  while  longer  filaments 
move  in  a  serpentine  manner. 

In  addition  to  the  volatile  fat  acids  which,  according  to  Brieger, 
are  formed  in  small  amounts  in  cultures  of  the  typhoid  bacillus,  and 
to  lactic  acid  formed  in  solutions  containing  grape  sugar,  a  basic 
substance  possessing  toxic  properties  has  been  isolated  by  the  chemist 
named — his  typhotoxine  (C,H17!N"03).  Brieger  supposes  that  other 
basic  substances  are  likewise  formed,  but  believes  this  to  be  the  speci- 
fic product  to  which  the  pathogenic  action  of  the  bacillus  is  due.  It 
is  a  strongly  alkaline  base,  which  produces  in  mice  and  guinea-pigs 
salivation,  paralysis,  dilated  pupils,  diarrhoea,  and  death. 

Numerous  experiments  have  been  made  to  determine  the  amounts 
of  various  germicidal  agents  required  to  destroy  the  vitality  of  this 
bacillus,  and  the  action  of  antiseptics  in  restraining  its  development. 
For  the  results  of  these  experiments  the  reader  is  referred  to  the 
sections  in  Part  Second  relating  to  the  action  of  antiseptics  and  disin- 
fectants. 

Pathogenesis. — The  very  numerous  experiments  which  have  been 
made  on  the  lower  animals  have  not  been  successful  in  producing  in 
any  one  of  them  a  typical  typhoid  process.  Nor  is  this  surprising, 
in  view  of  the  fact  that,  so  far  as  is  known,  no  one  of  them  is  liable  to 
contract  the  disease,  as  man  does,  by  the  use  of  infected  food  or 
water. 

The  experiments  of  Frankel  and  Simmonds  show  that  when  con- 
siderable quantities  of  a  pure  culture  of  this  bacillus  are  injected  into 
the  circulation  of  rabbits  through  the  ear  vein,  or  into  the  peritoneal 
cavity  of  mice,  a  certain  proportion  of  the  inoculated  animals  die, 


THE   BACILLUS   OF   TYPHOID    FEVER. 


443 


usually  within  forty-eight  hours,  and  that  the  bacillus  may  be  re- 
covered from  the  various  organs,  although  it  is  not  present  in  the 
blood.  But  death  does  not  always  occur  from  intravenous  injections, 
and  subcutaneous  or  intraperitoneal  injections  in  rabbits  are  usually 
without  result.  Subcutaneous  injections  in  mice  proved  to  be  fatal  in 
ten  cases  out  of  sixteen  inoculated  by  A.  Frankel.  Seitz,  by  following 
Koch's  method — i.  e. ,  by  rendering  the  contents  of  the  stomach  alka- 
line, and  arresting  intestinal  peristalsis  by  the  administration  of 
opium — obtained  a  fatal  result,  in  a  majority  of  the  guinea-pigs  experi- 
mented upon,  from  the  introduction  of  ten  cubic  centimetres  of  a 
bouillon  culture  into  the  stomach  through  a  pharyngeal  catheter. 
We  may  remark,  with  reference  to  these  results,  that  while  they  show 
that  cultures  of  the  typhoid  bacillus  have  a  certain  pathogenic  power, 


FIG.  111.— Section  through  wall  of  intestine,  showing  invasion  by  typhoid  bacilli.    X  950. 
(Baumgarten.) 

they  also  show  that  the  animals  experimented  upon  frequently  re- 
covered after  comparatively  large  doses,  and  that  the  typhoid  bacil- 
lus is  not  pathogenic  in  the  same  sense  as  are  those  microorganisms 
which,  when  introduced  into  the  body  of  a  susceptible  animal  in  very 
minute  amount,  give  rise  to  general  infection  and  death.  On  the 
other  hand,  a  fatal  result  depends  upon  the  quantity  of  the  culture 
introduced  in  the  first  instance,  rather  than  upon  the  multiplication 
of  the  bacillus  in  the  body  of  the  inoculated  animal.  This  view  is 
confirmed  by  the  experiments  of  Sirotinin,  which  show  not  only  that 
a  fatal  result  depends  upon  the  quantity  injected,  but  also  that  a 
similar  result  follows  the  injection  of  cultures  which  have  been  ster- 
ilized by  heat  or  filtration.  The  pathogenic  action,  then,  depends 
upon  the  presence  of  toxic  substances  produced  during  the  growth  of 
the  bacillus  in  artificial  culture  media.  The  researches  of  Brieger, 
heretofore  referred  to,  show  the  presence  in  such  cultures  of  a  toxic 
ptomaine — his  typhotoxine — to  which  the  pathogenic  potency  of  these 


444  THE   BACILLUS   OF   TYPHOID    FEVER. 

cultures  appear  to  be  due.  White  mice  and  guinea-pigs  usually  die 
in  from  twenty-four  to  forty-eight  hours  when  inoculated  in  the 
cavity  of  the  abdomen  with  a  virulent  culture  of  the  typhoid  bacillus 
—0.1  cubic  centimetre  to  0.5  cubic  centimetre  of  a  bouillon  culture 
three  days  old.  According  to  Kitasato,  the  virulence  of  cultures 
from  different  cases  of  typhoid  fever  varies  considerably. 

Detection  of  the  Typhoid  Bacillus  in  Water. — The  generally 
recognized  fact  that  typhoid  fever  is  usually  contracted  by  drink- 
ing water  contaminated  by  the  typhoid  bacillus  has  led  to  numer- 
ous researches  having  for  their  object  the  discovery  of  a  reliable 
method  of  detecting  this  bacillus  when  present  in  water  in  compara- 
tively small  numbers  in  association  with  the  ordinary  water  bacilli. 
The  use  of  Koch's  plate  method,  as  commonly  employed,  will 
not  suffice,  because  the  water  bacilli  present  grow  more  rapidly 
and  cause  liquefaction  of  the  gelatin  before  visible  colonies  of  the 
typhoid  bacillus  are  formed  ;  and,  owing  to  the  relatively  small 
number  of  typhoid  bacilli,  these  are  likely  to  escape  detection.  The 
aim  of  bacteriologists  has,  therefore,  been  to  restrain  the  growth  of 
these  common  water  bacilli  by  some  agent  which  does  not  at  the 
same  time  prevent  the  development  of  the  typhoid  bacillus.  Chan- 
temesse  and  Widal  were  the  first  to  propose  the  use  of  carbolic  acid 
for  this  purpose.  They  recommended  the  addition  of  0. 25  per  cent 
of  this  agent  to  nutrient  gelatin  ;  but,  according  to  Kitasato,  the  de 
velopment  of  the  typhoid  bacillus  is  restrained  by  an  amount  exceed- 
ing 0.20  per  cent. 

Holz  prepares  an  acid  medium  by  adding  gelatin  (ten  per  cent)  to 
the  juice  of  raw  potatoes,  and  asserts  that  while  the  typhoid  bacillus 
grows  luxuriantly  in  this  medium,  many  other  bacilli  fail  to  develop 
in  it.  The  test  is  said  to  be  still  more  reliable  if  0.05  per  cent  of  car- 
bolic acid  is  added  to  the  "  potato-gelatin."  According  to  Holz,  the 
addition  of  more  than  0. 1  per  cent  of  carbolic  acid  to  nutrient  gelatin 
prevents  the  free  development  of  the  typhoid  bacillus. 

Thoinot  has  claimed  to  be  able  to  obtain  the  typhoid  bacillus  from 
mixed  cultures — as,  for  example,  from  f^ces — by  suspending  a  small 
amount  of  material  containing  it  for  several  hours  in  a  solution  con- 
taining 0.25  per  cent  of  carbolic  acid.  While  other  bacilli  are 
destroyed,  the  typhoid  bacillus  is  said  to  survive  such  exposure. 

The  method  of  Parietti  has  been  tested  in  a  practical  way  by 
Kamen,  and  proved  to  be  satisfactory  for  the  detection  of  the  typhoid 
bacillus  in  water  which  was  supposed  to  be  the  source  of  a  local 
epidemic  of  the  disease.  The  following  solution  is  used : 

Carbolic  acid,        ........         5  grammes. 

Hydrochloric  acid  (pure), 4 

Distilled  water, 100 


THE   BACILLUS   OF   TYPHOID    FEVER.  445 

Several  test  tubes,  each  of  which  contains  ten  cubic  centimetres 
of  neutral,  sterilized  bouillon,  are  used  in  the  experiment.  From 
three  to  nine  drops  of  the  acid  solution  are  added  to  each  of  these, 
and  the  tubes  are  then  placed  in  an  incubating  oven  for  twenty-four 
hours  to  ascertain  whether  they  are  still  sterile  after  this  addition. 
If  the  bouillon  remains  clear,  from  one  to  ten  drops  of  the  suspected 
water  are  added  to  each  tube  and  they  are  returned  to  the  incubating 
oven.  If  at  the  end  of  twenty-four  hours  the  bouillon  becomes 
clouded,  this  is  due,  according  to  Parietti,  to  the  presence  of  the 
typhoid  bacillus,  which  is  then  to  be  obtained  in  pure  cultures  by  the 
plate  method. 

The  following  method,  suggested  by  Hazen  and  White,  has  been 
tested  with  favorable  results  by  Foote.  This  method  depends  upon 
the  fact  that  most  of  the  common  water  bacilli  do  not  grow  at  a  tem- 
perature of  40°  C.,  whereas  this  is  a  favorable  temperature  for  the 
development  of  the  typhoid  bacillus.  A  small  quantity  of  the  sus- 
pected water  is  added  to  liquefied  nutrient  agar  in  test  tubes,  and 
plates  are  made.  These  are  placed  in  an  incubating  oven  at  40°  C., 
and  the  typhoid  bacillus,  if  present,  will  develop  colonies  within  two 
or  three  days.  At  the  ordinary  room  temperature  the  more  numerous 
water  bacilli  would  develop  upon  the  same  plates  so  abundantly  that 
it  would  be  difficult  to  recognize  colonies  of  the  typhoid  bacillus. 

Theobald  Smith  (Centralb.  f.  Bakteriol.,  Bd.  xii.,  page  367), 
has  shown  that  the  typhoid  bacillus  may  be  differentiated  from  other 
similar  bacilli  (Bacillus  coli  communis,  bacillus  of  hog  cholera,  etc.) 
by  the  fact  that  it  does  not  produce  gas  in  culture  media  containing 
sugar — grape  sugar,  cane  sugar,  or  milk  sugar.  The  medium  recom- 
mended by  Smith  for  making  this  test  is  a  peptone-bouillon  contain- 
ing two  per  cent  of  grape  sugar  and  made  slightly  alkaline  with 
carbonate  of  soda.  The  liquid  becomes  clouded  throughout  at  the 
end  of  twenty-four  hours,  but  not  a  trace  of  gas  is  developed  even 
after  several  days.  On  the  other  hand,  the  colon  bacillus  and  other 
bacilli  which  closely  resemble  the  typhoid  bacillus  cause  an  abundant 
development  of  gas  in  this  medium. 

The  method  of  Wurtz  will  be  found  useful  in  the  detection  of 
colonies  of  the  typhoid  bacillus  in  plate  cultures  from  contaminated 
water,  etc.  This  consists  in  the  addition  to  the  nutrient  medium  of 
lactose  (two  per  cent)  and  a  solution  of  litmus.  When  the  colonies 
develop  in  plates  made  from  this  medium  the  typhoid  colonies  re- 
main blue,  while  colonies  of  the  "  colon  bacillus  "  have  a  red  color,  on 
account  of  the  development  of  lactic  acid. 

Schild  (1894)  uses  a  bouillon  containing  formalin  (1:7,000)  and 
claims  that  the  typhoid  bacillus  fails  to  grow  in  this  medium,  while 


446  THE   BACILLUS   OF   TYPHOID   FEVER. 

the  bacilli  of  the  colon  group  multiply  in  it  and  cause  the  me- 
dium to  become  clouded  within  twenty-four  hours.  Abel  (1894),  as  a 
result  of  extended  experiments,  arrives  at  the  conclusion  that  the 
formalin  test  cannot  be  relied  upon  for  distinguishing  the  typhoid 
bacillus  from  certain  similar  bacilli,  which  also  fail  to  grow  in  for- 
malin solution.  •  But,  on  the  other  hand,  a  bacillus  which  grows  in 
bouillon  containing  1 : 7,000  of  formalin  can  be  definitely  pronounced 
to  be  not  the  typhoid  bacillus. 

Eisner  (1895)  recommends  the  following  method  for  the  detection 
of  the  typhoid  bacillus  in  water  or  in  faeces :  To  potato  gelatin,  pre- 
pared by  the  method  of  Holz,  he  added  one  per  cent  of  potassium 
iodide.  But  few  species  of  bacteria  will  grow  in  this  medium,  but 
Bacillus  coli  communis  grows  in  it  luxuriantly,  forming  fully  de- 
veloped colonies  at  the  end  of  twenty-four  hours.  The  typhoid  col- 
onies, on  the  contrary,  are  only  just  visible  under  a  low  power  at  the 
end  of  twenty-four  hours,  and  at  the  end  of  forty-eight  hours  are 
seen  as  small,  shining,  drop-like,  very  finely  granular  colonies.  At 
the  same  time  the  colonies  of  the  colon  bacillus  are  much  larger, 
coarsely  granular,  and  of  a  brownish  color.  By  this  method  Eisner 
succeeded  in  obtaining  pure  cultures  of  the  typhoid  bacillus  from 
the  fa3ces  in  fifteen  out  of  seventeen  cases  of  typhoid  fever,  in  various 
stages  of  the  disease.  Lazarus  (1895)  has  tested  this  method  and  re- 
ports that  he  succeeded  without  any  difficulty  in  obtaining  pure  cul- 
tures of  the  typhoid  bacillus  from  the  alvine  discharges  of  typhoid 
patients. 

When  the  typhoid  bacillus  and  the  colon  bacillus  are  planted  to- 
gether, in  the  same  liquid  medium,  the  first-mentioned  bacillus,  even 
when  in  excess  at  the  outset  of  the  experiment,  soon  disappears  and 
the  Bacillus  coli  communis  remains  in  full  possession.  According 
to  Wathelet  (1895)  the  colon  bacillus  will  grow  in  bouillon  which 
has  served  as  a  culture  medium  for  the  typhoid  bacillus,  or  on  the 
surface  of  an  agar  plate  from  which  a  typhoid  culture  has  been  re- 
moved; but  the  typhoid  bacillus  fails  to  develop  in  culture  media 
which  have  served  for  the  development  of  the  colon  bacillus. 

The  various  diagnostic  tests  which  have  been  proposed,  and  the 
extensive  literature  of  the  subject,  show  that  the  recognition  of  the 
typhoid  bacillus  in  water,  fasces,  etc.,  is  attended  with  serious  diffi- 
culties. This  is  chiefly  due  to  the  fact  that  bacilli  have  been  ob- 
tained from  various  sources  which  resemble  more  or  less  closely  the 
typical  typhoid  bacillus  as  obtained  from  the  spleen  of  a  typhoid 
patient  (or  cadaver)  and  the  "  colon  bacillus  "  as  found  in  the  alimen- 
tary canal  of  healthy  men  and  animals;  and  also  from  the  fact  that 
the  bacillus,  as  obtained  from  typhoid  cases,  varies  to  some  extent  in 
its  biological  characters,  and  that  varieties  may  be  produced  in  the 


THE   BACILLUS    OF   TYPHOID    FEVER.  44? 

bacillus  as  obtained,  from  a  single  colony,  by  special  modes  of  culti- 
vation. From  a  consideration  of  these  facts  certain  authors  have 
been  led  to  the  conclusion  that  Bacillus  typhi  abdominalis  and  Bacillus 
coli  communis  are  simply  varieties  of  the  same  species.  This  view, 
however,  is  not  generally  accepted,  and  the  characters  which  serve  to 
differentiate  the  two  bacilli  are  sufficiently  well  defined  when  typical 
cultures  are  compared.  These  characters,  briefly  stated,  are:  The 
invisible  growth  of  the  typhoid  bacillus  on  potato;  its  failure  to  give 
the  indol  reaction;  its  failure  to  coagulate  milk,  or  to  produce  a 
change  of  color  in  litmus  milk ;  its  failure  to  produce  gas  in  culture 
media  containing  glucose  or  lactose;  its  failure  to  grow  in  formalin 
bouillon  (1 : 7,000) ;  and  its  active  motility.  Whether  the  closely  re- 
lated bacilli  which  present  some  of  the  characters  above  indicated, 
without  corresponding  in  all  particulars  with  typical  cultures  of  the 
typhoid  bacillus,  are  varieties  of  this  bacillus,  which  under  favorable 
circumstances  could  give  rise  to  typhoid  infection,  has  not  been  defi- 
nitely determined,  but  appears  to  be  quite  probable.  It  may  be  that 
such  varieties  are  developed  when  the  typhoid  bacillus  in  faeces  finds 
its  way  into  surface  waters,  under  conditions  which  are  favorable  for 
its  continued  development  as  a  saprophyte.  On  the  other  hand,  it 
may  be  that  one  or  more  of  the  saprophytic  bacilli,  which  are  found 
in  water  and  which  closely  resemble  the  typhoid  bacillus,  may  give  rise 
to  the  infectious  disease  which  we  know  as  typhoid  fever  when  in- 
troduced into  the  alimentary  canal  of  a  particularly  susceptible  indi- 
vidual, and  that  the  special  conditions  attending  its  development  as 
a  parasite  give  rise  to  certain  modifications  in  its  biological  charac- 
ters of  a  more  or  less  permanent  kind. 

Frankland  (1895),  as  a  result  of  extended  experiments,  has  arrived 
at  the  conclusion  that  when  the  typhoid  bacillus  is  cultivated  for  a 
long  time  in  media  which  are  more  and  more  largely  diluted  with 
water,  it  acquires  an  increased  ability  to  survive  in  river  water. 

A  predisposition  to  typhoid  infection  is  established  -by  various 
depressing  agencies,  such  as  inanition,  overwork,  mental  worry,  in- 
sanitary surroundings,  etc.  And  there  is  considerable  evidence  in 
support  of  the  supposition  that  exposure  to  the  offensive  gases 
given  off  from  ill- ventilated  sewers  constitutes  a  predisposition  to 
the  disease. 

Experiments  made  by  Alessi  (1894),  in  the  Hygienic  Institute 
of  the  University  of  Rome,  give  support  to  this  view.  The  ex- 
periments were  made  upon  rats,  guinea-pigs,  and  rabbits.  The 
rats  were  confined  in  a  close  cage  with  perforated  bottom,  which  was 
placed  over  the  opening  of  a  privy;  the  guinea-pigs  and  rabbits  in 
similar  cages  having  a  receptacle  below  in  which  their  own  excreta 
was  allowed  to  accumulate.  The  animals  which  breathed  an  atmo- 


448  THE  BACILLUS   OF  TYPHOID   FEVER. 

sphere  vitiated  in  this  way  lost,  after  a  time,  their  usual  activity  and 
became  emaciated,  although  they  continued  to  eat  greedily.  When 
these  animals  were  inoculated  with  a  small  quantity  of  a  culture  of 
the  typhoid  bacillus  (0.25  to  0.5  cubic  centimetre)  they  died  within 
from  twelve  to  thirty-six  hours.  The  same  amount  of  the  typhoid 
culture  injected  into  control  animals  produced  no  injurious  effect.  In 
the  animals  which  succumbed  to  typhoid  infection  there  was  found  a 
hemorrhagic  enteritis,  incfease  in  volume  of  Peyer's  glands  and  of  the 
spleen,  and  typhoid  bacilli  in  the  blood,  liver,  and  spleen.  The  char- 
acteristic appearances  of  typhoid  infection  were  more  pronounced  in 
the  rabbits  and  guinea-pigs  than  in  rats.  Similar  experiments  with 
Bacillus  coli  communis  gave  similar  results.  The  time  required  to 
induce  this  predisposition  for  typhoid  infection  was  from  five  to 
seventy-two  days  for  the  rats,  seven  to  fifty-eight  for  the  guinea- 
pigs,  and  three  to  eighteen  for  the  rabbits.  Alessi  found  that  the 
susceptibility  to  infection  diminished  after  a  certain  time,  and  sug- 
gests that  in  a  similar  way  man  may  become  habituated  to  breathing 
an  atmosphere  containing  sewer  gases. 

Pus- Production  by  Typhoid  Bacilli. — The  literature  relat- 
ing to  the  typhoid  bacillus  includes  many  observations  as  to  its 
presence  in  accumulations  of  pus  in  various  parts  of  the  body — often 
in  a  pure  culture.  It  has  been  found  in  a  considerable  number  of 
cases  of  periostitis  secondary  to  typhoid  fever,  in  purulent  syno- 
vitis,  and  in  abscesses  in  various  parts  of  the  body. 

Dmochowski  and  Janowski  (1895),  as  the  result  of  a  review  of  the 
literature  and  a  painstaking  experimental  research,  arrive  at  the  con- 
clusion that  even  in  abscesses,  occurring  in  typhoid  fever  cases,  in 
which  only  the  pus  cocci  are  found,  it  is  probable  that  the  typhoid 
bacillus  originated  the  process  resulting  in  abscess  formation.  They 
assert  that  the  typhoid  bacillus  dies  out  in  a  comparatively  short 
time  in  abscesses  which  are  directly  due  to  its  presence,  and  that 
often  it  may  be  found  in  the  abscess  walls  when  its  presence  can  no 
longer  be  demonstrated  in  the  purulent  contents  of  the  abscess  cavity. 


PLATE   V. 

PATHOGENIC    BACTERIA. 

FIG.  1. — Bacillus  anthracis  from  cellular  tissue  of  inoculated  mouse. 
Stained  with  gentian  violet.  X  1,000.  Photomicrograph  by  Frankel 
and  Pfeiffer. 

FIG.  2. — Bacillus  anthracis  in  section  of  liver  of  inoculated  rabbit. 
Stained  with  Bismarck  brown,  x  250.  Photomicrograph  by  Sternberg. 

FIG.  3. — Micrococcus  gonorrhoeas  in  gonorrhceal  pus.  Stained  with 
gentian  violet,  x  1,000.  Photomicrograph  by  gaslight.  (Sternberg.) 

FIG.  4. — Anthrax  spores  from  a  bouillon  culture.  Double-stained 
preparation — with  carbol-f uchsin  and  rnethylene  blue,  x  1,000.  Photo- 
micrograph by  Frankel  and  Pfeiffer. 

FIG.  5. —  Spirillum  cholerse  Asiaticee  from  a  culture  upon  starched 
linen  at  end  of  twenty-four  hours.  Stained  with  f uchsin.  x  1,000. 
Photomicrograph  by  Frankel  and  Pfeiffer. 

FIG.  6. — Bacillus  diphtherise  from  colony  upon  an  agar  plate,  twenty- 
four  hours  old.  Stained  with  Loffler's  solution  of  methylene  blue,  x 
1,000.  Photomicrograph  by  Frankel  and  Pfeiffer. 


PLATE  V. 

STERNBERG'S  BACTERIOLOGY. 


igf.  2. 


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it 


PigT.  4. 


Pig.  6. 


Pig.  6. 


X. 
BACTERIA   IN   DIPHTHERIA. 

DIPHTHERIA  is  generally  recognized  by  physicians  as  a  specific 
infectious  disease,  and,  owing  to  its  wide  prevalence  and  fatal  char- 
acter, a  precise  knowledge  of  its  etiology  is  of  the  greatest  import- 
ance. Until,  as  a  result  of  recent  researches,  this  was  determined, 
pathologist s  were  in  doubt  as  to  whether  diphtheria  should  be  con- 
sidered as  primarily  a  local  infection,  or  whether  the  local  manifesta- 
tions were  secondary  to  a  general  systemic  infection.  But  this  question 
appears  now  to  be  definitely  settled  in  favor  of  the  former  view.  We 
have  to-day  a  very  precise  knowledge  of  the  specific  infecting  agent, 
and  have  evidence  that  it  produces  during  its  growth  a  very  potent 
toxic  substance,  the  absorption  of  which  from  the  seat  of  local  infec- 
tion accounts  in  a  satisfactory  manner  for  the  general  symptoms  of 
the  disease,  which  are  due  to  toxaemia  and  not  to  an  invasion  of  the 
blood  and  tissues  by  the  pathogenic  microorganism  producing  it. 

Numerous  researches  by  competent  bacteriologists  have  failed  to 
demonstrate  the  presence  of  bacteria  in  the  blood  of  patients  suffer- 
ing from  diphtheria,  but  a  variety  of  microorganisms  have  been  ob- 
tained in  cultures  from  diphtheritic  pseudo-membranes,  and  may  be 
demonstrated  by  the  microscopical  examination  of  stained  prepara- 
tions. Among  these  are  the  well-known  pus  organisms,  and  espe- 
cially the  Streptococcus  pyogenes,  which  appears  to  be  very  commonly 
present,  and  is  perhaps  the  active  agent  in  the  production  of  certain 
forms  of  pseudo-diphtheria.  But  the  malignant,  specific  diphtheria, 
so  well  known  in  this  country  and  in  Europe,  has  been  demonstrated 
by  the  recent  researches  of  bacteriologists  to  be  due  to  a  bacillus  first 
recognized  by  Klebs  in  stained  preparations  of  diphtheritic  false 
membranes  (1883),  and  cultivated  and  described  by  Lofner  in  1884. 
In  his  first  publication  Lofner  did  not  claim  to  have  fully  demon- 
strated the  etiological  relation  of  this  bacillus,  but  this  appears  to  be 
fully  established  by  subsequent  researches. 

In  his  first  research  Lofner  studied  twenty-five  cases,  and  in  the 
greater  number  of  them  found  in  stained  preparations  the  bacil- 
lus previously  described  by  Klebs.  From  six  of  these  cases  he 
29 


450  BACTERIA   IX   DIPHTHERIA. 

obtained  it  in  pure  cultures,  and  by  inoculations  in  pigeons,  chickens, 
rabbits,  and  guinea-pigs  proved  that  it  gave  rise  to  a  diphtheritic 
inflammation  when  inoculated  into  the  mucous  membrane  of  the 
trachea,  conjunctiva,  pharynx,  or  vagina.  In  a  second  communica- 
tion Loftier  reported  his  success  in  finding  the  same  bacillus  in  ten 
additional  cases,  and  also  that  he  had  isolated  from  the  same  source 
a  non-pathogenic  bacillus  which  resembled  it  very  closely.  This 
pseudo-diphtheria  bacillus  has  since  been  found  by  other  bacteri- 
ologists (Von  Hoffmann,  Roux  and  Yersin),  and  it  is  uncertain 
whether  it  is  to  be  considered  a  distinct  species,  or  a  non-pathogenic 
variety  of  the  diphtheria  bacillus  as  maintained  by  Roux  and  Yersin. 
But  its  occasional  presence  does  not  invalidate  the  very  positive  ex- 
perimental evidence  relating  to  the  specific  pathogenic  power  of  the 
true  diphtheria  bacillus. 

Loffler,  in  1890,  reviewed  the  evidence  upon  which  this  bacillus  is 
now  generally  conceded  by  bacteriologists  to  be  the  specific  infectious 
agent  in  true  diphtheria.  The  following  are  the  principal  points  in 
the  demonstration : 

FIRST. — It  is  found  in  all  undoubted  cases  of  diphtheria.  In 
support  of  this  we  have  the  results  of  researches  made  by  Loffler, 
Wyssokowitsch,  D'Espine,  Von  Hoffmann,  Ortmann,  Roux  and 
Yersin,  Kolisko  and  Paltauf,  Zarinko  and  Sorensen,  who  in  nearly 
every  case  have  demonstrated  without  difficulty  the  presence  of  this 
bacillus.  On  the  other  hand,  Prudden  failed  to  find  it  in  a  series  of 
twenty-four  cases  studied  by  him ;  but  his  own  account  of  these 
cases  indicates  that  they  were  not  cases  of  true  diphtheria.  He  says 
in  a  subsequent  communication  : 

4 'In  view  of  the  doubt  existing  among  practitioners  as  to  whether  all 
forms  of  pseudo-membranous  inflammation  should  be  called  diphtheria  or 
not,  and  with  the  purpose  of  making  a  wholly  objective  study,  the  writer 
distinctly  stated  at  the  outset  of  that  paper  that  all  the  fatal  cases  of  exten- 
sive pseudo-membranous  laryngitis,  as  well  as  pharyngitis,  should  in  his 
study  be  considered  as  cases  of  diphtheria.  This  left  the  question  as  to  the 
propriety  of  establishing  separate  groups  of  pseudo-membranous  inflamma- 
tion open  and  free  from  bias.  It  was  distinctly  stated,  however,  that  six- 
teen out  of  the  twenty-four  cases  occurred  in  a  large  asylum,  in  which 
measles  and  scarlet  fever  were  prevalent  during  the  period  in  which  these 
studies  were  under  way.  Five  other  cases  in  another  asylum  were  ex- 
posed to  similar  conditions." 

In  a  subsequent  series  of  "  twelve  cases  of  fatal  pseudo-mem- 
branous inflammation  occurring  in  two  children's  asylums,  in  which 
for  many  months  there  had  been  no  scarlatina  and  no  measles,  and 
in  which  there  was  no  complicating  suppurative  inflammation  and 
no  erysipelas,"  Prudden  (1890)  obtained  Loffler's  bacillus  in  cultures 
from  eleven,  and  he  says  : 

"We  are  now,  it  would  seem,  justified,  as  it  did  not  appear  to  the  writer 


BACTERIA   IN   DIPHTHERIA.  451 

that  we  were  two  years  ago,  owing  to  the  large  number  of  important  re- 
searches which  have  been  made  in  the  interim,  in  saying  that  the  name 
diphtheria,  or  at  least  primary  diphtheria,  should  be  applied,  and  exclusively 
applied,  to  that  acute  infectious  disease,  usually  associated  with  a  pseudo- 
membranous  inflammation  of  the  mucous  membranes,  which  is  primarily 
caused  by  the  bacillus  called  Bacillus  diphtherias  of  Loffler.'' 

With  reference  to  the  question  as  to  how  long  after  convalescence 
is  established  the  diphtheria  bacillus  may  be  present  in  the  throat 
of  an  infected  person,  Loffler  has  made  the  following  research  (1890). 
In  a  typical  case  a  bacteriological  examination  was  made  daily  from 
the  commencement  until  fourteen  days  after  its  termination.  Fever 
disappeared  on  the  fifth  day,  and  the  exudation  had  all  disappeared 
on  the  sixteenth  day.  Up  to  this  time  the  bacillus  was  daily  ob- 
tained in  cultures,  and  subsequently  nearly  every  day  up  to  the 
twenty-fifth — that  is,  for  three  weeks  after  the  febrile  symptoms  had 
disappeared.  Roux  and  Yersin  have  also  obtained  the  bacillus  in 
cultures  from  mucus  scraped  from  the  throats  of  convalescents  sev- 
eral days  after  the  disappearance  of  all  evidence  of  the  disease. 

SECOND.  The  Klebs-Loffler  bacillus  is  found  only  in  diph- 
theria.— In  his  earlier  researches  Loffler  obtained  the  bacillus  in  a 
single  instance  from  the  mouth  of  a  healthy  child,  and  this  fact  led 
him  to  hesitate  in  announcing  it  as  his  conviction  that  it  was  the 
true  cause  of  diphtheria.  But  in  extended  researches  made  subse- 
quently he  has  not  again  succeeded  in  finding  it,  except  in  associa- 
tion with  diphtheria,  and  admits  now  that  he  may  have  been  mis- 
taken as  to  the  identity  of  the  bacillus  found.  This  seems  not 
improbable  in  view  of  the  fact  that  very  similar  bacilli  have  been 
found  by  various  bacteriologists.  Thus  Von  Hoffmann  obtained  a 
very  similar  but  non-pathogenic  bacillus  from  the  mucus  of  chronic 
nasal  catarrh  and  from  healthy  mucous  membranes ;  Babes  from 
cases  of  trachoma,  Neisser  from  ulcers,  Zarinko  from  the  surface  of 
various  mucous  membranes.  But  all  of  these  were  shown  to  present 
certain  differences  in  their  biological  characters  by  which  they  could 
be  differentiated  from  the  true  diphtheria  bacillus. 

Welch  and  Abbott  in  their  comparative  studies  did  not  find  the 
Loffler  bacillus,  "or  any  bacillus  that  an  experienced  bacteriologist 
would  be  likely  to  confound  with  it."  They  examined  mucus  from 
the  throats  of  healthy  children,  from  those  suffering  from  simple  in- 
flammation of  the  tonsils  and  pharynx,  and  from  four  cases  of  so- 
called  follicular  tonsillitis.  As  a  result  of  their  investigations  they 
agree  with  Loffler,  and  with  Roux  and  Yersin,  as  to  "  the  great  prac- 
tical value,  for  diagnostic  purposes,  of  a  bacteriological  examination 
of  cover-glass  specimens  and  by  cultures  "  of  cases  in  which  there  is 
any  doubt  of  the  true  character  of  the  disease.  They  say  further  : 


452  BACTERIA   IN   DIPHTHERIA. 


i  i 


The  only  species  of  bacteria  which  we  have  found  constantly  in  the 
cases  of  diphtheria  has  been  the  Loffler  bacillus.  Two  other  species  have 
been  present  in  many  cases,  viz.,  the  well-known  streptococcus,  which  grows 
in  much  smaller  colonies  and  less  rapidly  than  the  Loftier  bacillus,  and  a 
short,  oval,  often  slightly  pointed  bacillus,  growing  in  long  chains  running 
parallel  to  each  other.  There  are  often  marked  irregularities  in  shape  and 
especially  in  size  of  this  bacillus,  even  of  individuals  in  the  same  chain. 
The  colonies  of  this  bacillus  are  grayish-white,  moist,  larger  than  those  of 
the  streptococcus,  but  smaller  than  those  of  the  Loffler  bacillus." 

THIRD.  As  shown  by  Loffler's  earlier  researches,  pure  cultures 
of  this  bacillus  induce  characteristic  diphtheritic  inflammation 
when  inoculated  into  the  mucous  membranes  of  certain  lower  ani- 
mals. Roux  and  Yersiii  have  also  shown  that  local  paralysis  is 
likely  to  occur  in  inoculated  animals,  as  is  the  case  in  diphtheria  in 
man.  In  speaking  of  their  inoculations  into  the  trachea  in  rabbits 
these  investigators  say  : 

"The  affection  which  is  thus  induced  in  the  rabbit  resembles  croup  in 
man.  The  difficulty  which  the  animal  experiences  in  breathing;  the  noise 
made  by  the  air  in  passing  through  the  obstructed  trachea-  the  aspect  of  the 
trachea,  which  is  congested  and  covered  with  false  membranes ;  the  oederna- 
tous  swelling  of  the  tissues  and  glands  of  the  neck,  make  the  resemblance 
absolutely  remarkable." 

Welch  and  Abbott  give  the  following  account  of  the  results  of 
inoculations  into  the  trachea  in  kittens  : 

"  A  half -grown  kitten  is  inoculated  into  the  trachea  with  one  platinum 
loop  from  a  pure  culture  of  the  Loffler  bacillus  on  glycerin-agar,  eleven  days 
old,  derived  from  Case  IV.  For  the  inoculation  a  small  median  incision  was 
made  over  the  trachea,  in  which  a  hole  just  large  enough  to  admit  the  plati- 
num loop  was  made.  The  culture  was  rubbed  over  the  mucosa  of  the  trachea 
for  an  extent  about  three  centimetres  in  length,  and  in  this  process  sufficient 
force  was  used  to  abrade  the  mucous  membrane.  On  the  day  following  the 
inoculation  no  special  alteration  in  the  animal  was  observed,  but  on  the 
morning  of  the  second  day  it  was  found  very  weak.  In  the  course  of  this 
day  it  became  so  weak  as  to  lie  completely  motionless,  apparently  uncon- 
scious, with  very  feeble,  shallow  respiration ;  several  times  it  was  thought  to 
be  dead,  but  on  careful  examination  proved  still  to  be  breathing  feebly.  It 
was  found  dead  on  the  morning  of  the  third  day.  At  the  autopsy  the  wound 
was  found  gaping  and  covered  with  a  grayish,  adherent,  necrotic,  distinctly 
diphtheritic  layer.  For  a  considerable  distance  around  the  wound  the  sub- 
cutaneous tissues  were  very  cedematous,  the  oedema  extending  from  the 
lower  jaw  down  over  the  sternum,  and  to  the  sides  of  the  neck,  and  along 
the  anterior  extremities.  The  lymphatic  glands  at  the  angle  of  the  jaw  were 
markedly  swollen  and  reddened.  The  mucous  membrane  of  the  trachea, 
beginningat  the  larynx  and  extending  down  for  six  centimetres,  wascovered 
with  a  tolerably  firm,  grayish-white,  loosely  attached  pseudo-membrane,  in 
all  respects  identical  with  the  croupous  membranes  observed  in  the  same 
situation  in  cases  of  human  diphtheria. " 


BACTERIA    IX    DIPHTHERIA.  453 

BACILLUS   DIPHTHERIA. 

First  observed  by  Klebs  (1883)  in  diphtheritic  false  membranes. 
Isolated  in  pure  cultures  and  pathogenic  power  demonstrated  by 
Loffler  (1884). 

Found  in  diphtheritic  pseudo-membranes,  and  especially  in  the 
deeper  portions,  intermingled  with  numerous  cellular  elements;  while 
the  superficial  layers  of  the  membrane  commonly  contain  but  few 
cells  or  bacilli,  or  are  invaded  by  other  species,  especially  by  Strep- 
tococcus pyogenes.  The  bacilli  are  not  found  in  the  affected  mucous 
membrane,  or  in  sections  from  the  internal  organs  in  fatal  cases  of 
this  disease. 

llor/tfiology. — Rods,  straight  or  slightly  curved,  with  rounded 
ends,  having  a  diameter  of  0.5  to  0.8 
ii.  and  from  I  to  3  /i  in  length.  Ir- 
regular forms  are  very  common,  and, 
indeed,  are  characteristic  of  this  bacil- 
lus. In  the  same  culture,  and  especially 
in  an  unfavorable  culture  medium,  very 
great  differences  in  form  and  dimen- 
sions may  be  observed  ;  one  or  both  ends 
may  appear  swollen,  or  the  central  por- 
tion may  be  notably  thicker  than  the 
extremities,  or  the  rod  may  be  made  up 
of  irregular  spherical  or  oval  segments.  FlG.  113>  _  Bacillus  diphtheria, 
Multiplication  occurs  by  fission  only,  from  a  culture  upon  blood  serum. 

I   ,T_      -I        -IT    i  ,    •  m  From  a  photomicrograph,     x  1,000. 

aild  the  bacilli  dO  not  grow  Out  into  fila-         (Frankel  and  Pfeiffer.) 

ments. 

In  unstained  preparations  certain  portions  of  the  rod,  and  espe- 
cially the  extremities,  are  observed  to  be  more  highly  refractive  than 
the  remaining  portion  ;  and  in  stained  preparations  these  portions 
are  seen  to  be  most  deeply  colored.  The  diphtheria  bacillus  may  be 
stained  by  the  use  of  Loffler 's  alkaline  solution  of  methylene  blue, 
but  is  not  so  readily  stained  with  some  of  the  other  aniline  colors 
commonly  employed.  It  stains  also  by  Gram's  method.  For  the 
demonstration  of  the  bacillus  in  sections  of  diphtheritic  membrane 
"  nothing  can  surpass  in  brilliancy  and  sharp  differentiation  sections 
stained  doubly  by  the  modified  Weigert's  fibrin  stain  and.  picro-car- 
mine"  (Welch  and  Abbott). 

Biological  Characters. — The  diphtheria  bacillus  is  aerobic,  non- 
motile',  and  non-liquefying;  it  does  not  form  spores.  It  grows  most 
freely  in  the  presence  of  oxygen,  but  is  also  a  facultative  anaerobic. 

Development  occurs  in  various  culture  media  at  a  temperature  of 
from  20°  to  42°  C.,  the  most  favorable  temperature  being  about  359  C. 


454  BACTERIA   IX   DIPHTHERIA. 

It  grows  readily  in  nutrient  gelatin  having  a  slightly  alkaline  reac- 
tion, in  nutrient  agar,  glycerin-agar,  or  in  alkaline  bouillon,  but  the 
most  favorable  medium  appears  to  be  that  first  recommended  by 

Loffler — viz.,  a  mixture  of  three 
parts  of  blood  serum  with  one  part 
;          of  bouillon,  containing  one  per  cent 
;f,t      of  peptone,  one  per  cent  of  grape 
sugar,  and  0. 5  per  cent  of  sodium 
chloride.     This  mixture  is  steril- 
ized and  solidified  at  a  low  tem- 
perature,  as  is   usual  with  blood 
serum.      Upon  this   the  develop- 
Fni.  us.— Colonies  of  Bacillus  diphtherias     meiit  is  so  rapid  in  the  incubating 

in  nutrient  agar,  end  of  twenty-four  hours.  ,,      ,        ,     ,,  -,       -  f  - 

X  10.    (FrankelandPfeiffer.)  lt>  at 

four  hours,  the  large,  round,  ele- 
vated colonies,  of  a  grayish-white  color  and  moist  appearance,  may 
be  easily  recognized,  while  other  associated  bacteria  will,  as  a  rule, 
not  yet  have  developed  colonies  large  enough  to  interfere  with  the 
recognition  of  these. 

Upon  nutrient  agar  plates  the  deep-lying  colonies,  when  magni- 
fied about  eighty  diameters,  appear  as  round  or  oval,  coarsely  granu- 
lar discs,  with  rather  ill-defined  margins,  or,  when  several  colonies 
are  in  juxtaposition,  as  figures  of  irregular  form.  The  superficial  col- 
onies are  grayish-yellow  in  color,  have  an  irregular,  not  well-defined 
outline  and  a  rough,  almost  reticulated  surface.  The  growth  upon 
glycerin-agar  is  very  similar.  The  first  inoculations  in  a  plain  nu- 
trient agar  tube  often  give  a  comparatively  feeble  growth,  which  be- 
comes more  abundant  in  subsequent  inoculations  in  the  same  medium. 
In  stick  cultures  in  glycerin — or  plain — agar,  growth  occurs  to  the 
bottom  of  the  line  of  inoculation,  and  also  upon  the  surface,  but  is 
not  at  all  characteristic.  The  same  may  be  said  with  reference  to 
cultures  in  nutrient  gelatin.  Plate  cultures  in  this  medium  contain- 
ing fifteen  per  cent  of  gelatin,  at  24°  C.,  give  rather  small  colonies, 
which  are  white  by  reflected  light  and  under  the  microscope  are  seen 
as  yellowish-brown,  opaque  discs,  having  a  more  or  less  irregular 
outline  and  a  granular  structure.  In  alkaline  bouillon  the  growth  is 
sometimes  in  the  form  of  small,  whitish  masses  along  the  sides  and 
bottom  of  the  tube,  but  at  others  a  diffusely  clouded  growth  occurs 
in  this  medium  ;  after  standing  for  some  time  in  the  incubating  oven 
a  thin,  white  pellicle  may  form  upon  the  surface  of  the  bouillon. 
The  reaction  of  the  bouillon  becomes  at  first  acid,  but  later  it  has  an 
alkaline  reaction  (Welch).  With  reference  to  the  growth  onpotato, 
authors  have  differed,  probably  because  the  growth  is  scarcely  vis- 
ible ;  upon  this  point  we  quote  from  Welch  and  Abbott  : 


BACTERIA   IN    DIPHTHERIA. 

"  Our  experience  has  been  that  the  Bacillus  diphtherias  grows  on  ordinary 
steamed  potato  without  any  preliminary  treatment,  but  that  the  growth  is 
usually  entirely  invisible  or  is  indicated  by  a  dry,  thin  glaze  after  several 
days.  Doubtless  the  invisible  character  of  the  growth  has  led  most  observers 
into  the  error  of  supposing  that  no  growth  existed,  whereas  the  microscopi- 
cal examination  reveals  a  tolerably  abundant  growth,  which  on  the  first  po- 
tato is  often  feebler  than  on  succeeding  ones.  Irregular  forms  are  par- 
ticularly numerous  in  potato  cultures,  and  in  general  the  rods  are  thicker 
than  on  other  media.  In  twenty-four  hours,  at  a  temperature  of  35°  C., 
microscopical  examination  shows  distinct  growth.  We  have  cultivated  the 
bacillus  for  many  generations  on  potato." 

Milk  is  a  favorable  medium  for  the  growth, of  this  bacillus,  and, 
as  it  grows  at  a  comparatively  low  temperature  (20°  C.),  it  is  evi- 
dent that  this  fluid  may  become  a  medium  for  conveying  the  bacillus 
from  an  infected  source  to  the  throats  of  previously  healthy  children. 

Cultures  of  the  diphtheria  bacillus  may  retain  their  vitality  for 
several  months,  and  when  dried  upon  silk  threads  for  several  weeks 
colonies  are  still  developed  in  a  suitable  medium — in  the  room  from 
three  to  four  weeks,  in  an  exsiccator  five  to  ten,  and  in  one  instance 
fourteen  weeks.  In  dried  diphtheritic  membrane,  preserved  in  small 
fragments,  the  bacillus  retained  its  vitality  for  nine  weeks,  and  in 
larger  fragments  for  twelve  to  fourteen  weeks. 

The  thermal  death-point,  as  determined  by  Welch  and  Abbott,  is 
58°  0.,  the  time  of  exposure  being  ten  minutes.  Loffler  had  previ- 
ously found  that  it  did  not  survive  exposure  for  half  an  hour  to  60° 
C.  With  reference  to  the  action  of  germicidal  and  antiseptic  agents, 
we  refer  to  the  sections  in  Part  Second  relating  to  this  subject. 

Pathogenesis. — In  view  of  the  evidence  heretofore  recorded,  it 
may  be  considered  as  demonstrated  that  this  bacillus  gives  rise  to 
the  morbid  phenomena  which  characterize  the  fatal  disease  in  man 
known  as  diphtheria. 

We  have  already  referred  to  the  effects  of  inoculations  into  the 
trachea  in  rabbits  and  cats,  which  give  rise  to  a  characteristic  diph- 
theritic inflammation,  with  general  toxaemia  and  death  from  the 
absorption  of  soluble  toxic  products  formed  at  the  seat  of  local  in- 
fection. This  inference  as  to  the  cause  of  death  seems  justified  by 
the  fact  that  the  pathogenic  bacillus  does  not  invade  the  blood  and 
tissues,  and  is  supported  by  additional  experimental  evidence  (see 
pages  309-317). 


PSEUDO-DIPHTHERITIC   BACILLUS. 

Loffler,  Von  Hoffmann,  and  others  have  reported  finding  bacilli 
which  closely  resemble  the  Bacillus  diphtheria?,  but  which  differ 
from  it  chiefly  in  being  non-pathogenic.  The  following  account  we 


456  BACTERIA   IN   DIPHTHERIA. 

take  from  a  paper  upon  the  subject  by  Roux  and  Yersiii  (troisieme 
memoire,  1890). 

Found  by  Roux  and  Yersin  in  mucus  from  the  pharynx  and  ton- 
sils of  children — from  forty-five  children  in  Paris  hospitals,  suffering 
from  various  affections,  not  diphtheritic,  fifteen  times;  from  fifty- 
nine  healthy  children  in  a  village  school  on  the  seaboard,  twenty-six 
times.  Of  six  children  with  a  simple  angina  but  two  furnished  cul- 
tures of  this  bacillus,  while  it  was  obtained  in  five  out  of  seven  ca.ses 
of  measles. 

Its  characters  are  given  as  follows  : 

'The  colonies  of  the  pse ml o -diphtheritic  bacillus,  cultivated  upon  blood 
serum,  are  identical  with  the  true  diphtheria  bacillus  At  a  temperature  of 
33°  to  35  multiplication  is  rapid,  and  it  continues  at  the  ordinary  tempera- 
ture, although  slowly.  Under  the  microscope  the  appearance  of  the  bacillus 
which  forms  these  colonies  is  the  same  as  that  of  Bacillus  diphtherige.  It 
stains  readily  with  Loffler's  solution  of  methylene  blue,  and  intensely  by 
Gram's  method.  Sometimes  it  colors  uniformly,  at  others  it  appears  granu- 
lar. It  grows  in  alkaline  bouillon,  giving  a  deposit  upon  the  walls  of  the 
vessel  containing  the  culture,  and  in  this  medium  often  presents  the  inflated 
forms,  pear-shaped,  or  club-shaped.  It  is  destroyed  in  a  liquid  medium  by  a 
temperature  of  58°  C.  maintained  for  ten  minutes.  All  of  these  characters 
are  common  to  the  pseudo-diphtheritic  bacillus  and  the  true  Bacillus  diphthe- 
riae.  As  a  difference  between  them  we  may  note  that  the  pseudo  diphtheritic 
bacillusds  often  shorter  in  colonies  grown  upon  blood  serum;  that  its  cultures 
in  bouillon  are  more  abundant ;  that  they  continue  at  a  temperature  of  20°  to 
22°,  at  which  the  true  bacillus  grows  very  slowly.  When  we  make  a  com- 
parison of  cultures  in  bouillon  they  become  acid  and  then  alkaline,  but  the 
change  occurs  much  sooner  in  the  case  of  the  pseudo-diphtheritic  bacillus. 
Like  the  true  bacillus,  the  pseudo  diphtheritic  grows  in  a  vacuum,  but  less 
abundantly  than  the  other. 

"  Inoculations  into  animals  of  cultures  of  this  bacillus  have  never  caused 
their  death ;  but  we  may  remark  that  in  some  experiments  a  notable  oedema 
has  been  produced  in  guinea-pigs  at  the  point  of  inoculation,  while  in  others 
there  has  been  no  local  lesion.  The  most  marked  oedema  resulted  from  cul- 
tures obtained  from  cases  of  measles. 

"  Do  the  facts  which  we  have  reported  explain  the  question  which  occupies 
us  ?  Can  we  conclude  that  there  is  a  relation  between  the  two  bacilli  ?  On 
the  one  side,  the  presence  of  the  pseudo  diphtheritic  bacillus  in  the  mouths  of 
healthy  persons,  and  of  those  who  have  anginas  manifestly  not  diphtheritic, 
seems  to  be  opposed  to  the  idea  of  a  relationship  between  them.  On  the 
other  hand,  when  we  consider  that  the  non- virulent  bacillus  is  very  rare  in 
fatal  diphtheria,  that  it  is  more  abundant  in  benign  diphtheria,  that  it  be- 
comes more  common  in  severe  cases  as  they  progress  towards  recovery,  and. 
finally,  that  they  are  more  numerous  in  persons  who  have  recently  had 
diphtheria  than  in  healthy  persons,  it  is  difficult  to  accept  the  idea  that  the 
two  microbes  are  entirely  distinct.  The  morphological  differences  which 
have  been  referred  to  are  so  slight  that  they  prove  nothing.  The  two  micro- 
organisms can  only  be  distinguished  by  their  action  upon  animals,  but  the 
difference  of  virulence  does  not  at  all  correspond  with  the  difference  of  ori- 

fin.  As  regards  the  form  and  the  aspect  of  cultures,  the  true  and  false 
iphtheria  bacilli  differ  less  than  virulent  anthrax  differs  from  a  very  attenu- 
ated anthrax  bacillus,  which,  however,  originate  from  the  same  source. 
Besides,  the  sharp  distinction  which  we  make  between  the  virulent  and  11011- 
virulent  bacilli  is  arbitrary ;  it  depends  upon  the  susceptibility  of  guinea- 
pigs.  If  we  inoculate  animals  still  more  susceptible,  there  are  pseudo  diph- 
theritic bacilli  which  we  must  class  as  virulent;  and  if,  on  the  contrary,  we 
substitute  rabbits  for  guinea  pigs  in  our  experiments,  there  are  diphtheritic 
bacilli  which  we  must  call  pseudo-diphtheritic.  In  our  experiments  \vo  do 


BACTERIA    IN   DIPHTHERIA.  457 

not  simply  encounter  bacilli  which  are  very  virulent  and  bacilli  which  are 
non- virulent;  between  these  two  extremes  there  are  bacilli  of  every  degree 
of  virulence." 


Abbott,  in  1891,  published  the  result  of  his  researches  with 
reference  to  the  presence  of  the  pseudo-diphtheritic  bacillus  in 
benign  throat  affections.  He  made  a  bacteriological  study  of  fifty- 
three  patients,  nine  of  whom  were  suffering  from  acute  phaiyngitis, 
fourteen  from  acute  follicular  tonsillitis,  eight  from  ordinary  post- 
nasal  catarrh,  two  from  simple  enlarged  tonsils,  fifteen  from  chronic 
pharyngitis,  one  from  subacute  laryngitis,  one  from  chronic  laryngi- 
tis, one  from  rhinitis,  and  two  from  an  affection  of  the  tonsils  and 
pharynx.  In  forty-nine  cases  nothing  of  particular  interest  was  ob- 
served. A  variety  of  microorganisms  were  isolated,  and  of  these 
the  pyogenic  micrococci  were  the  most  common. 

In  four  cases  microorganisms  were  found  which  resembled  the 
Bacillus  diphtheria  of  Loffler  in  their  morphology  and  growth  in  cul- 
ture media,  but  which  proved  not  to  be  pathogenic.  Abbott  says  : 
"  The  single  point  of  distinction  that  can  be  made  out  between  the 
organisms  obtained  from  Cases  L,  III.,  and  IV.  and  the  true  bacil- 
lus of  diphtheria  is  in  the  absence  of  pathogenic  properties  from  the 
former,  whereas  in  addition  to  this  point  of  distinction  the  organism 
from  Case  II.  gives,  as  has  been  stated,  a  decided  and  distinct 
growth  upon  the  surface  of  sterilized  potato. " 

Recent  authors  are  generally  inclined  to  the  opinion  that  bacilli 
which  resemble  the  diphtheria  bacilli  in  every  respect  except  that 
they  are  non-pathogenic  should  be  regarded  as  attenuated  varieties 
of  the  diphtheria  bacillus  rather  than  as  belonging  to  a  distinct 
species — the  so-called  "pseudo-diphtheria"  bacillus.  However,  there 
are  bacilli  which  closely  resemble  the  bacillus  of  diphtheria  and  yei 
may  be  differentiated  from  it  otherwise  than  by  the  test  upon  sus- 
ceptible animals.  Neisser  has  given  us  a  staining  method  which  is 
especially  useful  in  making  this  differential  diagnosis.  The  culture 
of  the  bacillus  to  be  tested  is  grown  upon  Loifler's  blood-serum  mix- 
ture. This  is  solidified  at  a  temperature  of  100°  C.,  and  grown  in 
an  incubator  at  a  temperature  between  34°  and  36°  C.  The  staining 
of  a  cover-glass  preparation  from  such  a  culture  is  effected  by  the 
following  method :  Methylene  blue,  one  gramme;  alcohol  (96°),  two 
cubic  centimetres ;  dissolve  and  add  distilled  water,  nine  hundred  and 
fifty  cubic  centimetres,  and  acetic  acid,  fifty  cubic  centimetres.  From 
one  to  three  seconds  only  will  be  required  to  stain  the  cover-glass 
preparation  with  this  solution;  it  should  then  be  carefully  washed  in 
water  and  stained  in  a  solution  made  by  adding  two  grammes  of 
vesuvin  to  one  litre  of  boiling  water.  This  solution  is  allowed  to 
cool  before  using,  and  from  three  to  five  seconds  will  be  sufficient 


458  BACTERIA   IN   DIPHTHERIA. 

time  for  the  action  of  the  stain,  after  which  the  cover  glass  is  again 
washed  and  is  then  ready  for  examination.  The  diphtheria  bacillus 
appears  in  such  a  preparation  as  faintly  stained  brown  rods,  in  the 
interior  of  which  one  to  three  dark-blue  granules  may  be  seen.  These 
are  oval  in  form  and  are  found  at  the  extremities  of  the  bacterial 
cells.  Neisser  and  others  who  have  made  use  of  this  method  agree 
that  bacilli  which  do  not  stain  in  this  way  are  not  diphtheria  bacilli. 

BACILLUS    DIPHTHERIA   COLUMBARUM. 

Described  by  Loffler  (1884),  who  obtained  it  from  diphtheritic  pseudo-mem- 
branes in  the  mouths  of  pig-eons  dead  from  an  infectious  form  of  diphtheria 
which  prevails  in  some  parts  of  Germany  among  these  birds  and  among 
chickens. 

Reddened  patches  first  appear  upon  the  mucous  membrane  of  the  mouth 
and  fauces,  and  these  are  covered  later  with  a  rather  thick,  yellowish  layer 
of  fibrinous  exudate.  In  pigeons  the  back  part  of  the  tongue,  the  fauces, 
and  the  corners  of  the  mouth  are  especially  affected ;  in  chickens  the  tongue, 
the  gums,  the  nares,  the  larynx,  and  the  conjunctival  mucous  membrane. 
The  disease  is  especially  fatal  among  chickens,  the  young  fowls  and  those  of 
choice  varieties  being  most  susceptible.  It  is  attended  at  the  outset  by  fever, 
and  usually  proves  fatal  within  two  or  three  weeks,  but  may  last  for  several 
months. 

Morphology. — Short  bacilli  with  rounded  ends,  usually  associated  in  ir- 
regular masses,  and  resembling  the  bacilli  of  rabbit  septicaBmia  (fowl 
cholera),  but  a  little  longer  and  not  quite  so  broad.  In  sections  from  the 
liver  they  are  seen  in  irregular  groups  in  the  interior  of  the  vessels. 

Biological  Characters  — An  aerobic,  non-motile,  non-liquefying  bacillus. 

Grows  in  nutrient  gelatin  in  the  form  of  spherical,  white  colonies  along 
the  line  of  puncture,  and  upon  the  surface  as  a  whitish  layer.  Under  the 
microscope  the  colonies  in  gelatin  plates  have  a  yellowish-brown  color  and 
a  slightly  granular  surface.  Upon  blood  serum  the  growth  consists  of  a 
semi-transparent,  grayish- white  layer.  Upon  potato  SL  thin  layer  is  formed 
having  a  grayish  tint. 

Pathogenesis. — Pigeons  inoculated  with  a  pure  culture  in  the  mucous 
membrane  of  the  mouth  are  affected  exactly  as  are  those  which  acquire  the 
disease  naturally.  Subcutaneous  inoculations  in  pigeons  give  rise  to  an  in- 
flammation resulting  in  local  necrotic  changes.  Pathogenic  for  rabbits  and 
for  mice.  Subcutaneous  injections  in  mice  give  rise  to  a  fatal  result  in  about 
five  days.  The  bacillus  is  found  in  the  blood  and  in  the  various  organs,  in 
the  interior  of  the  vessels,  and  sometimes  in  the  interior  of  the  leucocytes ; 
they  are  especially  numerous  in  the  liver.  The  lungs  are  dotted  with  red 
spots,  the  spleen  is  greatly  enlarged,  and  the  liver  has  a  marbled  appearance 
from  the  presence  of  numerous  irregular  white  masses  scattered  through  the 
pale-red  parenchyma  of  the  organ.  These  white  masses  are  seen,  in  sec- 
tions, to  consist  of  necrotic  liver  tissue,  in  the  centre  of  which  the  bacilli 
are  found  in  great  numbers,  in  the  interior  of  the  vessels.  This  appearance 
is  so  characteristic  that  Loffler  considers  inoculations  in  mice  to  be  the  most 
reliable  method  of  establishing  the  identity  of  the  bacillus.  Not  pathogenic 
for  chickens,  guinea-pigs,  rats,  or  dogs. 

There  seems  to  be  some  doubt  whether  the  form  of  diphtheria  which  pre- 
vails among  pigeons,  and  which  Loffler  has  shown  to  be  due  to  the  bacillus 
above  described,  is  identical  with  the  diphtheria  of  chickens.  Diphtheria  in 
man  has  been  supposed  by  some  authors  to  be  identical  with  that  which 
prevails  among  fowls,  and  possibly  this  may  be  the  case  under  certain  cir- 
cumstances. But  the  evidence  seems  to  be  convincing  that  there  is  an 


BACTERIA   IN   DIPHTHERIA.  459 

infectious  diphtheria  of  fowls  which  is  peculiar  to  them,  and  which,  under 
ordinary  circumstances,  is  not  communicated  to  man. 

BACILLUS   DIPHTHERIAS   VITULORUM. 

Described  by  Loffler  (1884)  and  obtained  by  him  from  the  pseudo-mem- 
branous exudation  in  the  mouths  of  calves  suffering-  from  an  infectious  form 
of  diphtheria.  The  disease  is  characterized  by  the  appearance  of  yellow 
patches  upon  the  mucous  membrane  of  the  cheeks,  the  gums,  the  tongue, 
and  sometimes  of  the  larynx  and  nares  of  infected  animals.  There  is  a  yel- 
lowish discharge  from  the  nose,  an  abundant  flow  of  saliva,  occasional  at- 
tacks of  coughing,  and  diarrhoea.  Death  may  occur  at  the  end  of  four  or 
five  days,  but  usually  the  animal  survives  for  several  weeks.  Diphtheritic 
patches  similar  to  those  in  the  mouth  are  also  found  in  the  large  intestine, 
and  scattered  abscesses  in  the  lungs. 

Loffler,  in  a  series  of  seven  cases  examined,  obtained  from  the  deeper  por- 
tions of  the  pseudo-membranous  deposit  a  long  bacillus  which  appears  to  be 
the  cause  of  the  disease. 

Morphology. — Bacilli,  five  to  six  times  as  long  as  broad,  usually  united  in 
long  filaments.  The  diameter  of  the  rods  is  about  half  that  of  the  bacillus 
of  malignant  oedema. 

Biological  Characters.—  Attempts  to  cultivate  this  bacillus  in  nutrient 
gelatin,  blood  serum  from  sheep,  and  various  other  media  were  unsuccessful. 
But  when  fragments  of  tissue  containing  the  bacillus  were  placed  in  blood 
serum  from  the  calf  a  whitish  border,  consisting  of  the  long  bacilli,  was  de- 
veloped. These  could  not,  however,  be  made  to  grow  when  transferred  to 
fresh  blood  serum. 

Pathogenesis. —  Mice  inoculated  subcutaneously  with  the  fresh  diph- 
theritic exudation  died  in  from  seven  to  thirty  days.  The  autopsy  disclosed 
an  extensive  infiltration  of  the  entire  walls  of  the  abdomen,  which  often  pene- 
trated the  peritoneal  cavity  and  enveloped  the  liver,  the  kidneys,  and  the 
intestine  in  a  yellowish  exudate.  The  bacillus  was  found  in  this  exudate, 
and  by  inoculating  a  little  of  it  into  another  animal  of  the  same  species  a 
similar  result  was  obtained.  Not  pathogenic  for  rabbits  or  guinea-pigs. 

BACILLUS   OF   INTESTINAL  DIPHTHERIA   IN   RABBITS. 

Described  by  Ribbert  (1887)  and  obtained  \>y  him  from  the  organs  of  rab- 
bits which,  succumbed  to  an  affection  characterized  by  a  diphtheritic  inflam- 
mation of  the  mucous  membrane  of  the  intestine.  The  autopsy  revealed  also 
swelling  of  the  mesenteric  glands  and  minute  iiecrotic  foci  in  the  liver  and 
spleen. 

Morphology. — Bacilli  with  slightly  rounded  ends,  from  three  to  four  /* 
long  and  1  to  1.4  u.  in  diameter;  often  united  in  pairs  or  in  filaments  con- 
taining several  elements. 

Stains  with  the  aniline  colors,  but  not  so  readily  in  sections  as  some 
other  microorganisms.  Ribbert  recommends  staining  with  aniline-water- 
fuchsin  solution,  washing  in  water,  then  placing  the  sections  in  methylene 
blue  solution,  and  decolorizing  in  alcohol.  Does  not  stain  by  Gram's 
method. 

Biological  Characters. — An  aerobic,  non- liquefy  ing  (non -motile  ?)  ba- 
cillus. Upon  gelatin  plates  semi-transparent,  grayish  colonies  are  formed, 
which  later  have  a  brownish  color;  the  surface  of  these  is  finely  granular 
and  of  a  pearly  lustre.  In  stick  cultures  in  nutrient  gelatin  the  growth 
along  the  line  of  puncture  is  very  scanty.  On  potato  a  flat,  whitish  layer  is 
formed,  which  extends  slowly  over  the  surface.  Grows  best  at  a  temperature 
of  30  to  35°  C. 

Pathogenesis. — Pure  cultures  injected  into  the  peritoneal  cavity  or  sub- 
cutaneously in  rabbits  caused  the  death  of  these  animals  in  from  three  to 
fourteen  days,  according  to  the  quantity  injected.  At  the  autopsy  necrotic 


460  BACTERIA   IX   DIPHTHERIA. 

foci  are  found  in  the  liver  and  spleen,  and  the  niesenteric  glands  are  en- 
larged, but  the  intestine  presents  a  healthy  appearance.  But  when  cultures 
are  introduced  into  the  alimentary  canal  the  characteristic  diphtheritic  in- 
flammation of  the  mucous  membrane  of  the  intestine  is  induced.  This  re- 
sult was  obtained  both  by  direct  injection  into  the  lumen  of  the  intestine 
and  by  injecting  cultures  into  the  mouth. 

Additional  Notes  upon  Diphtheria  and  the  Diphtheria  Bacil- 
lus.— C.  Frankel  (1895)  reports  that  he  has  repeatedly  observed 
branching  forms  of  the  diphtheria  bacillus  in  cultures  upon  LCf- 
fler's  blood-serum  medium,  and  that  these  branching  forms  are  se<jn 
more  constantly  and  in  greater  numbers  in  cultures  made  upon  the 
surface  of  hard-cooked  albumen  from  hen's  eggs. 

The  continued  presence  of  virulent  diphtheria  bacilli  in  the  fauces 
of  patients  who  have  recovered  from  the  disease,  either  after  the  u^e 
of  the  antitoxin  or  under  other  treatment,  has  been  demonstrated  by 
several  bacteriologists.  Silverschmidt  (1895),  in  forty-five  cases 
treated  by  Behring's  antitoxic  serum,  found  that  the  number  of  ba- 
cilli usually  diminished  some  days  after  the  treatment  was  com- 
menced, but  that  in  cases  in  which  complete  recovery  had  taken 
place  not  infrequently  virulent  bacilli  could  be  obtained  many  days 
(in  one  case  thirty-one  days)  after  convalescence  was  established. 

Escherich  (1893)  opposes  the  view  that  the  pseudo-diphtheria  bacil- 
lus is  simply  a  non- virulent  variety  of  the  diphtheria  bacillus.  He 
found  this  pseudo-diphtheria  bacillus  in  the  throats  of  thirteen  out 
of  three  hundred  and  twenty  individuals  examined.  According  to 
him  there  is  no  evidence  that  this  completely  non-virulent  pseudo- 
diphtheria  bacillus  ever  acquires  pathogenic  virulence,  while  attenu- 
ated varieties  of  the  true  diphtheria  bacillus  readily  recover  their 
power  to  produce  the  toxic  products  upon  which  virulence  depends. 

Sevestre  (1895),  as  a  result  of  researches  made  by  himself  and 
several  other  bacteriologists  who  have  made  similar  investigations, 
arrives  at  the  conclusion  that : 

"  First.  In  a  certain  number  of  cases  the  bacillus  of  Loftier  disap- 
pears about  the  same  time  as  the  false  membranes;  or  it  may  persist 
for  some  time,  but  ceases  to  be  virulent — in  this  case  it  seems  to  have 
undergone  modifications  and  presents  the  form  of  short  bacilli.  .  .  . 

"  Second.  In  another  series  of  cases,  less  numerous  but  neverthe- 
less considerable,  the  bacillus  persists  in  a  virulent  condition  for  a 
longer  or  shorter  time  after  the  apparent  cure  of  the  malady.  .  .  . 

"  Third.  The  observations  collected  up  to  the  present  time  do  not 
enable  us  to  fix  precisely  the  limits  of  persistence,  but  it  is  not  far 
out  of  the  way  if  we  place  it  at  several  weeks  to  a  month  for  the 
throat.  In  the  nasal  fossaa  the  bacillus  often  persists  for  a  still 
longer  time,  and  its  presence  commonly  coincides  with  a  more  or  less 
abundant  discharge  from  the  nose." 


BACTERIA    IN    DIPHTHERIA.  461 

Park  and  Beebe  (1894),  in  an  extended  research  made  for  the  pur- 
pose of  determining  the  persistence  of  the  diphtheria  bacillus  in  the 
throats  of  convalescents  ('2,500  cultures  made),  found  that  in  304  out 
of  005  consecutive  cases  the  bacillus  disappeared  within  3  days  after 
the  disappearance  of  the  exudate;  in  170  cases  it  persisted  for  7  days; 
in  64  cases  for  12  days;  in  36  cases  for  15  days;  in  13  cases  for  3 
weeks;  in  4  cases  for  4  weeks;  in  2  cases  for  9  weeks.  Park  and 
Beebe  arrive  at  the  following  conclusion  with  reference  to  pseudo- 
diphtheria  bacilli : 

u  The  name  pseudo-diphtheria  bacillus  should  be  regarded  as  ap- 
plying to  those  bacilli  found  in  the  throat  which,  though  resembling 
the  diphtheria  bacilli  in  many  respects,  yet  differ  in  others  equally  im- 
portant. These  bacilli  are  rather  short,  and  more  uniform  in  size 
and  shape  than  the  typical  Loffler  bacillus.  They  stain  equally 
throughout  with  the  alkaline  methyl-blue  solution,  and  produce 
alkali  in  their  growths  in  bouillon.  They  are  found  in  about  one 
per  cent  of  the  healthy  throats  in  New  York  City,  and  seem  to  have 
no  connection  with  diphtheria.  They  are  never  virulent." 

Park  (1894)  has  shown  that  virulent  diphtheria  bacilli  are  fre- 
quently found  in  the  throats  of  persons  who  have  been  associated 
with  diphtheria  patients,  although  no  manifestations  of  the  disease 
were  visible.  It  is  therefore  apparent  that  infection  requires  not 
only  the  presence  of  virulent  bacilli,  but  also  of  a  predisposition  to 
the  disease.  This  corresponds  with  the  facts  relating  to  other  in- 
fectious diseases — e.g.,  tuberculosis,  typhoid  fever — and  among  the 
probable  predisposing  causes  we  may  mention  "  sewer-gas  poisoning," 
catarrhal  inflammations  of  the  mucous  membranes  most  commonly 
involved,  inanition,  "crowd  poisoning,"  and  depressing  agencies 
generally. 

Bacteriologists  have  given  much  attention  to  the  question  of  mixed 
infection  in  diphtheria.  Funck  (1894)  accepts  the  generally  received 
view  that  mixed  infections  with  the  diphtheria  bacillus  and  Strepto- 
coccus pyogenes  are  more  serious  than  an  uncomplicated  diphtheria, 
and  in  an  experimental  research  has  attempted  to  determine  whether 
this  is  due  to  an  increased  production  of  the  diphtheria  bacillus  or  to 
the  presence  of  the  streptococcus.  His  experiments  on  guinea-pigs 
showed  that  when  infected  with  streptococci  these  animals  did  not 
prove  to  be  more  sensitive  to  the  action  of  the  diphtheria  poison 
(without  living  bacilli),  and  he  concludes  that  the  unfavorable  influ- 
ence of  the  streptococcus  in  mixed  infections  is  due  to  increased  patho- 
genic activity  on  the  part  of  the  diphtheria  bacillus.  Bernheim 
(1894)  found,  in  his  experiments  on  guinea-pigs,  that  they  suc- 
cumbed more  rapidly  to  diphtheria  infection  when  they  previously 


402  BACTERIA   IN   DIPHTHERIA. 

or  simultaneously  received  an  injection  of  a  streptococcus  culture — 
filtered  or  unfiltered. 

Eesults  of  Treatment  ivith  the  Antitoxin. — While  questions  re- 
lating to  therapeutics  are  not  considered  in  this  manual,  a  brief  note 
upon  the  results  of  treatment  by  the  serum  of  immunized  animals 
may  not  be  out  of  place.  A  collective  investigation  (1895)  un- 
dertaken by  the  Deutsche  medicinische  Wochenschrift  gave  the 
following  results:  The  number  of  cases  collected  was  10,312;  all  of 
these  occurred  between  the  1st  of  October,  1894,  and  the  1st  of  April, 
1895;  5;883  of  these  cases  were  treated  with  the  antitoxin  and  4,479 
without  it.  In  the  first  group  the  mortality  was  9.6  per  cent,  and  in 
the  second  group  14.7  per  cent.  Two  thousand  five  hundred  and  fifty 
six  children  treated  with  the  antitoxin  were  between  two  and  ten 
years  of  age;  among  these  the  mortality  was  4  per  cent,  while 
among  children  of  the  same  age  not  treated  with  the  antitoxin  the 
mortality  was  15.2  per  cent.  Six  hundred  and  ninety-six  patients 
above  ten  years  of  age  were  treated  with  a  mortality  of  1  per  cent. 

Monod  (1895),  at  a  meeting  of  the  Paris  Academy  of  Medicine, 
presented  the  following  statistics  demonstrating  the  influence  upon 
the  mortality  from  diphtheria  in  France  exerted  by  the  antitoxin 
since  its  employment  from  November,  1894.  The  following  figures 
represent  the  number  of  deaths  from  diphtheria  during  the  first  six 
months  in  eight  years  in  108  French  cities  having  a  population  of 
more  than  20,000: 

1888-94.  1895. 

Average.         Average. 

January 469  205 

February 466  187 

March 499  155 

April 442  160 

May..     417  113 

June 333  84 

2,656  904 

It  will  be  seen  from  the  above  statement  that  during  the  first  six 
months  in  the  year  1895  after  the  introduction  of  the  antitoxin  treat- 
ment, the  number  of  deaths  from  diphtheria  in  the  108  French  cities 
referred  to  was  1,552  less  than  the  average  for  the  preceding  ten 
years,  and  we  are  justified  in  concluding  that  a  considerable  propor- 
tion of  this  saving  at  least  is  due  to  this  method  of  treatment. 


XI. 

BACILLUS  OF  INFLUENZA. 

DISCOVERED  by  Pfeiffer  (1892)  in  the  purulent  bronchial  secretion, 
and  by  Canon  in  the  blood  of  patients  suffering  from  epidemic  in- 
fluenza. Pfeiffer  found  the  bacillus  in  thirty-one  cases  examined  by 
him,  and  in  uncomplicated  cases  it  was  present  in  the  purulent  bron- 
chial secretion  in  immense  numbers  and  in  a  pure  culture.  Canon, 
whose  independent  observations  were  published  at  the  same  time, 
examined  the  blood  of  twenty  influenza  patients  in  stained  prepara- 
tions, and  found  the  same  bacillus  in  nearly  all  of  them.  His  method 
of  demonstrating  it  is  as  follows  : 

The  blood  is  spread  upon  clean  glass  covers  in  the  usual  way. 
After  the  preparations  are  thoroughly  dry  they  are  placed  in  abso- 
lute alcohol  for  five  minutes.  They  are  then  transferred  to  the  fol- 
lowing staining  solution  (Czenzynke's) :  concentrated  aqueous  solu- 
tion of  methylene  blue,  forty  grammes  ;  one-half -per-cent  solution  of 
eosin  (dissolved  in  seventy-per-cent  alcohol),  twenty  grammes  ;  dis- 
tilled water,  forty  grammes.  The  cover  glasses  immersed  in  this 
staining  solution  are  placed  in  an  incubating  oven  at  37°  C.  for  from 
three  to  six  hours,  after  which  they  are  washed  with  water,  dried, 
and  mounted  in  balsam.  In.  successful  preparations  the  red  blood 
corpuscles  are  stained  red  by  the  eosin,  and  the  leucocytes  blue.  The 
bacillus  is  seen  in  these  as  a  short  rod,  often  resembling  a  diplococcus. 
It  is  sometimes  seen  in  large  numbers,  but  usually  only  a  few  rods 
are  seen  after  a  long  search — four  to  twenty  in  a  single  preparation. 
In  six  cases  it  was  found  in  numerous  aggregations  containing  from 
five  to  fifty  bacilli  each.  In  these  cases  the  blood  was  drawn  during 
a  fall  of  temperature  or  shortly  after. 

^Morphology. — Very  small  bacilli,  having  about  the  same  diameter 
as  the  bacillus  of  mouse  septicaemia,  but  only  half  as  long.  Solitary 
or  united  in  chains  of  three  or  four  elements. 

Stains  with  difficulty  with  the  basic  aniline  dyes — best  with 
dilute  Ziehl's  solution,  or  Loffler's  methylene  blue  solution,  with  heat. 
The  two  ends  of  the  bacilli  are  most  deeply  stained,  causing  them  to 
resemble  diplococci.  Pfeiffer  says:  "I  am  inclined  to  believe  that 
some  of  the  earlier  observers  also  saw  the  bacilli  described  by  me, 
but  that,  misled  by  their  peculiar  behavior  with  regard  to  staining 
agents,  they  described  them  as  diplococci  or  streptococci/'  Do  not 
stain  by  Gram's  method. 


4G4  BACILLUS   OF   INFLUENZA. 

Biological  Characters. — An  aerobic,  non-motile  bacillus.  Does 
not  grow  in  nutrient  gelatin  at  the  room  temperature.  Spore  forma- 
tion not  observed.  Upon  the  surface  of  glycerin-agar  in  the  incubat- 
ing oven  very  small,  transparent,  drop-like  colonies  are  developed  at 
the  end  of  twenty-four  hours.  These  can  only  be  recognized  by  the 
aid  of  a  lens.  "  A  remarkable  point  about  them  is  that  the  colonies 
always  remain  separate  from  each  other,  and  do  not,  as  all  other 
species  known  to  me  do,  join  together  and  form  a  continuous  row. 
This  feature  is  so  characteristic  that  the  influenza  bacilli  can  be 
thereby  with  certainty  distinguished  from  other  bacteria"  (Kitasato). 
On  1.5  per  cent  sugar-agar  the  colonies  appear  as  extremely  small 
droplets,  clear  as  water,  often  only  recognizable  with  a  lens 
(Pfeiffer). 

In  bouillon  a  scanty  development  occurs,  and  at  the  end  of  twen- 
ty-four hours  small,  white  particles  are  seen  upon  the  surface,  which 
subsequently  sink  to  the  bottom,  forming  a  white,  woolly  deposit, 
while  the  bouillon  above  remains  transparent.  This  bacillus  does 
not  grow  at  temperatures  below  28°  0. 

Canon  has  obtained  colonies,  resembling  those  described  by  Kita- 
sato, in  cultures  from  the  blood  of  influenza  patients.  His  cultures 
were  made  upon  glycerin-agar  in  Petri's  dishes.  Ten  or  twelve  drops 
of  blood  from  a  puncture  made  in  the  finger  of  the  patient,  after 
sterilization  of  the  surface,  were  allowed  to  fall  upon  the  agar  medium, 
and  this  was  placed  in  the  incubating  oven.  As  the  number  of  ba- 
cilli in  the  blood  is  small,  a  considerable  quantity  is  used.  The 
colonies  are  visible  at  the  end  of  twenty-four  to  forty-eight  hours. 

The  influenza  bacillus  is  quickly  destroyed  by  desiccation  ;  a 
pure  culture  diluted  with  water  and  dried  is  destroyed  with  cer- 
tainty in  twenty  hours ;  in  dried  sputum  the  vitality  is  retained 
somewhat  longer,  but  no  growth  occurs  after  forty  hours.  The 
thermal  death-point  is  60°  C.  with  five  minutes'  exposure  (Pfeiffer 
and  Beck). 

Pathogenesis. — Pfeiffer  infers  that  this  is  the  specific  cause  of 
influenza  in  man  for  the  following  reasons  : 

1.  They  were  found  in  all  uncomplicated  cases  of  influenza  ex- 
amined, in  the  characteristic  purulent  bronchial  secretion,  often  in 
absolutely  pure  cultures.     They  were  frequently  situated  in  the  pro- 
toplasm of  the  pus  corpuscles  ;   in  fatal  cases  they  were  found  to 
have  penetrated  from  the  bronchial  tubes  into  the  peribronchitic  tis- 
sue, and  even  to  the  surface  of  the  pleura,  where  in  two  cases  they 
were  found  in  pure  cultures  in  the  purulent  exudation. 

2.  They  were  only  found  in  cases  of  influenza.     Numerous  con- 
trol  experiments  proved   their   absence    in   ordinary   bronchial   ca 
tarrh,  etc. 


PLATE   VI. 

PATHOGENIC    BACTERIA. 

FIG.  1. — Bacillus  of  influenza  in  bronchial  niucus.  x  1,000.  Photo- 
micrograph by  Frankel, 

FIG.  2. — Bacillus  of  influenza  in  bronchial  mucus,  after  the  termina- 
tion of  the  febrile  period.  The  bacilli  are  for  the  most  part  in  pus  cells. 
X  1,000.  Photomicrograph  by  Frankel. 

FIG.  3. — Bacillus  tetani  from  an  agar  culture.  x  1,000.  Photo- 
micrograph by  Frankel  and  Pfeiffer. 

FIG.  4. — Micrococcus  pneumonias  crouposse  in  sputum  of  a  patient 
with  pneumonia,  x  1,000.  Stained  by  Gram's  method.  Photomicro- 
graph by  Frankel  and  Pfeiffer. 

FIG.  5. — Micrococcus  pneumonise  crouposse  in  blood  of  rabbit,  x 
1,000.  Photomicrograph  made  at  the  Army  Medical  Museum,  Washing- 
ton, by  Gray. 

FIG.  6. — Bacillus  of  hog  cholera,  showing  flagella.  Stained  by 
Loffler's  method.  X  1,000.  Photomicrograph  made  at  the  Army  Medi- 
cal Museum,  Washington,  by  Gray. 


BACILLUS   OF   INFLUENZA.  465 

3.  The  presence  of  the  bacilli  corresponded  with  the  course  of  the 
disease,  and  they  disappeared  with  the  cessation  of  the  purulent 
bronchial  secretion. 

In  his  preliminary  report  of  his  investigations  Pfeiffer  says  : 

"  Numerous  inoculation  experiments  were  made  on  apes,  rabbits, 
guinea-pigs,  rats,  pigeons,  and  mice.  Only  in  apes  and  rabbits 
could  positive  results  be  obtained.  The  other  species  of  animals 
showed  themselves  refractory  to  influenza/' 

Kruse  (1894)  reports  that  he  found  the  bacillus  of  Pfeiffer  in 
eighteen  influenza  patients  examined  by  him  in  the  hospital  at  Bonn. 
On  the  other  hand,  he  failed  to  find  it  in  a  considerable  number  of  pa- 
tients suffering  from  other  diseases  of  the  respiratory  passages.  His 
evidence  is  the  more  valuable  as  he  had  previously  (1890)  reported 
his  failure  to  find  the  bacillus  in  typical  cases  of  influenza.  He  now 
ascribes  his  failure  at  that  time  to  imperfect  technique. 

Huber  (1893),  Richter  (1894),  Borchardt  (1894),  and  other  com- 
petent bacteriologists,  have  also  confirmed  the  results  reported  by 
Pfeiffer  as  regards  the  presence  of  this  bacillus  in  the  bronchial 
secretions  of  persons  suffering  from  epidemic  influenza,  and  as  to 
its  biological  characters.  Bujwid  (1893)  recognizes  the  bacillus  of 
Pfeiffer  as  identical  with  a  bacillus  which  he  cultivated  from  the 
spleen  of  an  influenza  patient  in  1890. 

The  researches  of  Weichselbaum,  Kowalski,  Friedrich,  Kruse, 
Bouchard,  and  others  have  given  a  negative  result  as  regards  the 
presence  of  the  influenza  bacillus  in  the  blood.  They  were  not  able 
to  demonstrate  its  presence  either  in  stained  preparations  or  by  cul- 
ture methods.  Pfeiffer,  also,  during  the  last  epidemic,  has  made 
special  researches  upon  this  point  and  has  never  succeeded  in  finding 
the  bacillus.  Day  after  day,  both  in  mild  and  severe  cases,  he  placed 
from  ten  to  twenty  drops  of  blood  from  influenza  patients  on  blood- 
agar — a  most  favorable  medium — but  his  cultures  always  remained 
sterile. 

In  his  experiments  upon  rabbits,  Pfeiffer  (1893)  found  that  the 
intravenous  injection  of  a  small  quantity  of  culture  on  blood-agar, 
twenty-four  hours  old,  suspended  in  one  cubic  centimetre  of  bouillon, 
caused  a  characteristic  pathogenic  effect.  The  first  symptoms  were 
developed  within  one  and  a  half  to  two  hours  after  the  injection. 
The  animals  became  extremely  feeble,  lying  flat  upon  the  floor  with 
their  limbs  extended,  and  suffered  from  extreme  dyspnoea.  The  tem- 
perature mounted  to  41°  C.  or  above.  At  the  end  of  five  or  six  hours 
thejr  were  able  to  sit  upon  their  haunches  again,  and  in  twenty-four 
hours  had  nearly  recovered  from  all  indications  of  ill-health.  Larger 
doses  caused  the  death  of  the  inoculated  animals.  These  results  are 
due  to  toxic  products  present  in  the  cultures,  and  Pfeiffer  has  never 
30 


466  BACILLUS   OF   INFLUENZA. 

observed  a  septicsemic  infection  as  a  result  of  his  inoculation  ex- 
periments. 

Pfeiffer  has  found  in  three  cases  of  bronchopneumonia  a  pseudo- 
influenza  bacillus  which  closely  resembles  the  bacillus  previously  de- 
scribed by  him  as  peculiar  to  that  disease.  This  pseudo-influenza 
bacillus  resembles  the  genuine  one  in  its  growth  in  culture  media, 
but  is  larger  and  shows  a  decided  inclination  to  grow  out  into  long 
threads.  By  these  morphological  characters,  which  are  said  to  be 
constant,  it  may,  according  to  Pteiffer,  be  readily  distinguished. 


XII. 
BACILLI  IN  CHRONIC  INFECTIOUS  DISEASES. 

IN  tuberculosis,  leprosy,  glanders,  and  syphilis  we  have  a  group 
of  infectious  diseases  which  present  many  points  of  resemblance. 
All  run  a  chronic  course  ;  all  may  be  communicated  to  susceptible 
animals  by  inoculation  ;  in  all,  the  lymphatic  glands  in  the  vicinity 
of  the  point  of  inoculation  become  enlarged,  and  new  growths,  con- 
sisting of  various  cellular  elements  of  a  low  grade  of  vitality,  are  de- 
veloped in  the  tissues  which  are  the  point  of  predilection  for  each  ; 
in  all,  these  new  growths  show  a  tendency  to  degenerative  changes, 
as  a  result  of  which  abscesses,  caseous  masses,  or  open  ulcers  are 
formed. 

In  two  of  the  diseases  in  this  group — tuberculosis  and  glan- 
ders— the  infectious  agent  has  been  obtained  in  pure  cultures  and  its 
specific  pathogenic  power  demonstrated  by  inoculations  in  susceptible 
animals;  in  one — leprosy — there  is  but  little  doubt  that  the  bacillus  con- 
stantly found  in  the  new  growths  characteristic  of  the  disease  bears 
an  etiological  relation  to  it,  although  this  has  not  been  demonstrated, 
the  bacillus  not  having  as  yet  been  cultivated  in  artificial  media. 
The  evidence  with  reference  to  the  parasitic  nature  of  the  fourth  dis- 
ease mentioned  as  belonging  to  this  group — syphilis — is  still  unsatis- 
factory, but  there  is  every  reason  to  believe  that  it  will  also  eventu- 
ally be  proved  to  be  due  to  a  parasitic  microorganism. 

The  announcement  of  the  discovery  of  the  tubercle  bacillus  was 
made  by  Koch,  in  March,  1882,  at  a  meeting  of  the  Physiological 
Society  of  Berlin.  At  the  same  time  satisfactory  experimental  evi- 
dence was  presented  as  to  its  etiological  relation  to  tuberculosis  in 
man  and  in  the  susceptible  lower  animals,  and  its  principal  biologi- 
cal characters  were  given. 

Baumgarten  independently  demonstrated  the  presence  of  the  tu- 
bercle bacillus  in  tuberculous  tissues  and  published  the  fact  soon 
after  the  appearance  of  Koch's  first  paper.  The  previous  demonstra- 
tion by  Villemin  (1865) — confirmed  by  Cohnheim  (1877)  and  others — 
that  tuberculosis  might  be  induced  in  healthy  animals  by  inocula- 
tions of  tuberculous  material,  had  paved  the  way  for  his  discovery, 


468  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

and  advanced  pathologists  were  quite  prepared  to  accept  it.  The 
more  conservative  have  since  been  obliged  to  yield  to  the  experi- 
mental evidence,  which  has  received  confirmation  in  all  parts  of  the 
world.  To-day  it  is  generally  recognized  that  tuberculosis  is  a  spe- 
cific infectious  disease  due  to  the  tubercle  bacillus. 

As  evidence  of  the  thorough  nature  of  Koch's  personal  researches 
in  advance  of  his  first  public  announcement,  we  give  the  following 
resume  of  his  investigations  : 

In  nineteen  cases  of  miliary  tuberculosis  the  bacilli  were  found  in 
the  tubercular  nodules  in  every  instance  ;  also  in  twenty-nine  cases 
of  pulmonary  phthisis,  in  the  sputum,  in  fresh  cheesy  masses,  and  in 
the  interior  of  recently  formed  cavities ;  in  tuberculous  ulcers  of  the 
tongue,  tuberculosis  of  the  uterus,  testicles,  etc.  ;  in  twenty-one  cases 
of  tuberculous — scrofulous — lymphatic  glands ;  in  thirteen  cases  of 
tuberculous  joints  ;  in  ten  cases  of  tubercular  bone  affections  ;  in  four 
cases  of  lupus  ;  in  seventeen  cases  of  Perlsucht  in  cattle.  His  ex- 
perimental inoculations  were  made  upon  two  hundred  and  seventy- 
three  guinea-pigs,  one  hundred  and  five  rabbits,  forty-four  field 
mice,  twenty-eight  white  mice,  nineteen  rats,  thirteen  cats,  and  upon 
dogs,  pigeons,  chickens,  etc.  Very  extensive  comparative  researches 
were  also  made,  which  convinced  him  that  the  bacillus  which  he  had 
been  able  to  demonstrate  in  tuberculous  sputum  and  tissues  by  a  spe- 
cial mode  of  staining  was  not  to  be  found  in  the  sputa  of  healthy 
persons,  or  of  those  suffering  from  non-tubercular  pulmonary  affec- 
tions, or  in  organs  and  tissues  involved  in  morbid  processes  of  a 
different  nature. 

BACILLUS   TUBERCULOSIS. 

Discovered  by  Koch  (first  public  announcement  of  discovery 
March  24th,  1882).  The  bacilli  are  found  in  the  sputum  of  persons 
suffering  from  pulmonary  or  laryngeal  tuberculosis,  either  free  or  in 
the  interior  of  pus  cells  ;  in  miliary  tubercles  and  fresh  caseous 
masses,  in  the  lungs  or  elsewhere  ;  in  recent  tuberculous  cavities  in 
the  lungs ;  in  tuberculous  glands,  joints,  bones,  and  skin  affections 
(lupus) ;  in  the  lungs  of  cattle  suffering  from  pulmonary  tubercu- 
losis— Perlsucht ;  and  in  tubercular  nodules  generally  in  animals 
which  are  infected  naturally  or  by  experimental  inoculations. 

In  the  giant  cells  of  tubercular  growths  they  have  a  peculiar  and 
characteristic  position,  being  found,  as  a  rule,  upon  the  side  of  the 
cell  opposite  to  the  nuclei,  which  are  crowded  together  in  a  crescentic 
arrangement  at  the  opposite  pole  of  the  cell.  Sometimes  a  single 
bacillus  will  be  found  in  this  position,  or  there  may  be  several. 
Again,  numerous  bacilli  may  be  found  in  giant  cells  in  which  the 
nuclei  are  distributed  around  the  periphery.  They  are  more  numer- 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  469 

ous  in  tuberculous  growths  of  recent  origin,  and  often  cannot  be 
demonstrated,  by  microscopical  examination,  in  caseous  material 
from  the  centre  of  older  nodules.  But  such  material,  when  inocu- 
lated into  susceptible  animals,  gives  rise  to  tuberculosis,  and  the 
usual  inference  is  that  it  contains  spores  of  the  tubercle  bacillus. 

Morphology. — The  tubercle  bacilli  are  rods  with  rounded  ends, 
of  from  1.5  to  3.5  ^  in  length,  and  are  commonly  slightly  curved  or 
bent  at  an  angle ;  the  diameter  is 
about  0.2  //.  In  stained  preparations 
unstained  portions  are  frequently 
seen,  which  are  generally  believed  to 
be  spores,  but  this  is  by  no  means 
certain.  From  two  to  six  of  these 
unstained  spaces  may  often  be  seen 
in  a  single  rod,  and  owing  to  this  al- 
ternation of  stained  and  unstained 
portions  the  bacilli  may,  under  a  low 
power,  be  mistaken  for  chains  of  mi- 
crococci  The  rods  are  usually  soli- 
tary, but  may  be  united  in  pairs,  or 

.      V  i      •  •    •  *  Fj°-      m-  —  Bacillus      tuberculosis. 

m  short  chains  containing  three  or  four     x  1,000.   From  a  photomicrograph, 
elements.     In   old  cultures  irregular 

forms  may  be  observed,  the  rods  being  sometimes  swollen  at  one 
extremity,  or  presenting  the  appearance  of  having  a  lateral  bud-like 
projection — involution  forms. 

The  staining  characters  of  this  bacillus  are  extremely  important 
for  its  differentiation  and  recognition  in  preparations  of  sputum,  etc. 
Unlike  most  microorganisms  of  the  same  class,  it  does  not  readily 
take  up  the  aniline  colors,  and  when  stained  it  is  not  easily  decolorized, 
even  by  the  use  of  strong  acids.  The  failure  to  observe  it  in  tuber- 
culous material,  prior  to  Koch's  discovery,  was  no  doubt  due  to  the 
fact  that  it  does  not  stain  in  the  usual  aqueous  solutions  of  the  aniline 
dyes.  Koch  first  recognized  it  in  preparations  placed  in  a  staining 
fluid  to  which  an  alkali  had  been  added — solution  of  methylene  blue 
with  caustic  potash  ;  but  this  method  was  not  very  satisfactory,  and 
he  promptly  adopted  the  method  devised  by  Ehrlich,  which  consists 
essentially  in  the  use  of  a  solution  of  an  aniline  color — fuchsin  or 
methyl  violet — in  a  saturated  aqueous  solution  of  aniline  oil,  and  de- 
colorization  with  a  solution  of  a  mineral  acid — nitric  acid  one  part  to 
three  parts  of  water. 

The  original  method  of  Ehrlich  gives  very  satisfactory  results, 
but  various  modifications  have  since  been  proposed,  some  of  which 
are  advantageous.  The  carbol-fuchsin  solution  of  Ziehl  is  now 
largely  employed ;  it  has  the  advantage  of  prompt  action  and  of 


470  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

keeping  well.  The  staining  is  effected  more  quickly  if  heat  is  ap- 
plied. The  tubercle  bacilli  stain  by  Gram's  method,  but  this  is  not 
to  be  recommended  for  general  use,  owing  to  the  fact  that  the  pro- 
toplasm of  the  rods  is  frequently  contracted  into  a  series  of  spheri- 
cal, stained  bodies,  which  might  easily  be  mistaken  for  micrococci. 

The  examination  of  sputum  for  the  presence  of  the  tubercle  ba- 
cillus is  recognized  as  a  most  important  procedure  for  the  early  diag- 
nosis of  pulmonary  tuberculosis.  It  is  at- 
tended with  no  special  difficulties,  and  every 
physician  should  be  acquainted  with  the 
technique. 

The  patient  should  be  directed  to  expec- 
torate into  a  clean,  wide-mouthed  bottle  or 
glass-covered  jar  the  material  coughed  up 
from   the  lungs,  and   especially,  in  recent 
cases,  that  which  is  coughed  up  upon  first 
rising    in   the   morning.     This    should   be 
FIQ.  us.— Bacillus  tubercuio-      placed  in  the  physician's  hands  as  promptly 
s^n  sputum,  x  i.ooo.  (Baum-      as  possibie  ;  although  a  delay  of  some  days 

does  not  vitiate  the  result,  and  the  tubercle 

bacilli  may  still  be  demonstrated  after  the  sputum  has  undergone  pu- 
trefaction. It  is  well  to  pour  the  specimen  into  a  clean,  shallow  vessel 
having  a  blackened  bottom — a  Petri's  dish  placed  upon  a  piece  of  dead- 
black  paper  will  answer  very  well.  In  tuberculous  sputum  small,  len- 
ticular masses  of  a  yellowish  color  may  usually  be  observed,  and  one 
of  these  should  be  selected  for  microscopical  examination,  by  picking 
it  up  with  a  platinum  needle  and  freeing  it  as  far  as  possible  from 
the  tenacious  mucus  in  which  it  is  embedded.  If  such  masses  are 
not  recognized  take  any  purulent-looking  material  present  in  the 
specimen,  whether  it  be  in  small  specks  distributed  through  the  mu- 
cus, or  in  larger  masses.  A  little  of  the  selected  material  should  be 
placed  in  the  centre  of  a  clean  cover  glass  and  another  thin  glass 
cover  placed  over  it.  By  pressure  and  a  to-and-fro  motion  the  mate- 
rial is  crushed  and  distributed  as  evenly  as  possible  ;  the  glasses  are 
then  separated  by  a  sliding  motion.  The  film  is  permitted  to  dry  by 
exposure  in  the  air.  When  dry  the  cover  glass,  held  in  forceps,  is 
passed  three  times  through  the  flame  of  an  alcohol  lamp  or  Bun  sen 
burner  to  fix  the  albuminous  coating.  Too  much  heat  causes  the  film 
to  turn  brown  and  ruins  the  preparation.  The  staining  fluid  (ZiehFs 
carbol-f  uchsin)  may  then  be  poured  upon  the  cover  glass,  or  this  may 
be  floated  upon  the  surface  of  the  fluid  contained  in  a  shallow  watch 
glass.  Heat  is  now  applied  by  bringing  the  cover  glass  over  a 
flame  and  holding  it  there  until  steam  begins  to  be  given  off  from 
the  surface  of  the  staining  fluid ;  it  is  then  withdrawn  and  again 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  4-71. 

gently  heated  at  intervals  for  a  minute  or  two.  The  cover  glass  is 
then  washed  in  water,  and  the  film  will  be  seen  to  have  a  uniform 
deep-red  color.  The  next  step  consists  in  decolorization  in  the  acid 
solution  (twenty-five-per-cent  solution  of  nitric  or  of  sulphuric  acid). 
The  cover  glass  is  gently  moved  about  in  this  solution  for  a  few 
seconds,  and  the  color  will  be  seen  to  quickly  fade  to  a  greenish 
tint.  The  object  is  to  remove  all  color  from  the  cells  and  the  al- 
buminous background,  so  that  the  bacilli,  which  retain  their  color  in 
presence  of  the  acid,  may  be  clearly  seen.  The  preparation  is  next 
washed  in  dilute  alcohol  (sixty  per  cent)  to  remove  the  fuchsin 
which  has  been  set  free  by  the  acid.  If  decolorization  was  not  car- 
ried far  enough  the  film  will  be  seen  to  still  have  a  red  color,  espe- 
cially in  places  where  it  is  thickest,  when  it  is  removed  from  the 
dilute  alcohol  and  washed  out  in  water.  In  this  case  it  will  be 
necessary  to  return  it  to  the  acid  solution  and  again  wash  it  in  the 
dilute  alcohol  and  in  water.  It  may  now  be  placed  in  a  solution 
of  methylene  blue  or  of  vesuvin  for  a  contrast  stain.  The  tubercle 
bacilli  are  distinguished  by  the  fact  that  they  retain  the  red  color 
imparted  to  them  in  the  fuchsin  solution,  while  other  bacteria  pre- 
sent, having  been  decolorized  in  the  acid  solution,  take  the  contrast 
stain  and  appear  blue  or  brown,  according  to  the  color  used.  The 
double-stained  preparation,  after  a  final  washing  in  water,  may  be 
examined  at  once,  or  dried  and  mounted  in  balsam  for  permanent 
preservation. 

Of  the  various  other  methods  which  have  been  proposed,  that  of 
Frankel,  as  modified  by  Gabbett,  appears  to  be  the  most  useful.  This 
consists  in  staining  as  above  directed  with  ZiehPs  carbol-f  uchsin  solu- 
tion, and  in  then  placing  the  cover  glass  directly  in  a  second  solution 
which  contains  both  the  acid  for  decolorizing  and  the  contrast  stain. 
This  second  solution  contains  twenty  parts  of  nitric  acid,  thirty  parts 
of  alcohol,  fifty  parts  of  water,  and  sufficient  methylene  blue  to  make 
a  saturated  solution  (one  to  two  parts  in  one  hundred).  After  re- 
maining in  this  solution  for  a  minute  or  two  the  cover  glas&  is  washed 
in  water,  and  upon  microscopical  examination  the  tubercle  bacilli,  if 
present,  will  be  seen  as  red  rods  which  strongly  contrast  with  the 
blue  background. 

The  methods  recommended  for  cover-glass  preparations  may  also 
be  used  for  staining  the  tubercle  bacillus  in  thin  sections  of  tuber- 
culous tissues,  except  that  it  is  best  not  to  employ  heat.  The  sec- 
tions may  be  left  for  an  hour  in  the  carbol-f  uchsin  solution,  or  for 
twelve  hours  in  the  Ehrlich-Weigert  tubercle  stain — eleven  cubic 
centimetres  of  saturated  alcoholic  solution  of  methyl  violet,  ten  cubic 
centimetres  of  absolute  alcohol,  one  hundred  cubic  centimetres  of  ani- 
line water.  They  should  then  be  decolorized  by  placing  them  for 


472 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


about  half  a  minute  in  dilute  nitric  acid  (ten  per  cent);  then  wash 
out  color  in  sixty-per-cent  alcohol ;  counter-stain  for  two  or  three 
minutes  in  a  saturated  aqueous  solution  of  methylene  blue  ;  dehydrate 
with  absolute  alcohol  or  with  aniline  oil ;  clear  up  in  oil  of  cedar, 
and  mount  in  xylol  balsam.  If  the  aniline-water-methyl-violet  solu- 
tion has  been  used  for  staining  the  bacilli  a  saturated  solution  of 
vesuvin  may  be  used  as  a  contrast  stain. 

Biological  Characters. — A  parasitic,  aerobic,  non-motile  ba- 
cillus, which  grows  only  at  a  temperature  of  about  37°  C.  Is  also  a 
facultative  anaerobic  (Frankel). 

The  question  as  to  spore  formation  has  not  been  definitely  deter- 
mined. It  has  been  generally  assumed  that  the  unstained  spaces 
which  are  frequently  seen  in  the  bacilli  are  spores  ;  and  the  fact  that 


FIG.  116.— Section  through  a  tuberculous  nodule  in  the  lung  of  a  cow,  showing  two  giant  cells 
containing  tubercle  bacilli.     X  950.     (Baumgarten.) 

caseous  material  in  which  a  microscopical  examination  has  failed  to 
demonstrate  the  presence  of  bacilli  may  produce  tuberculosis,  with 
bacilli,  when  inoculated  into  guinea-pigs,  has  been  explained  upon  the 
supposition  that  this  material  contained  spores.  But  a  few  bacilli 
present  in  such  caseous  material  might  easily  escape  detection.  As 
pointed  out  by  Frankel,  the  oval  spaces  in  stained  specimens  have 
not  the  sharply  defined  outlines  of  spores.  Moreover,  the  bacilli,  when 
examined  in  unstained  preparations,  do  not  contain  corresponding  re- 
fractive bodies,  recognizable  as  spores.  And  when  the  bacilli  are 
stained  by  Gram's  method  the  protoplasm  is  often  contracted  in  the 
form  of  little,  spherical  stained  masses,  while  the  unstained  spaces 
are  larger  and  no  longer  have  the  oval  form  presented  in  rods  stained 
by  Ehrlich's  method.  The  great  resisting  power  of  the  bacillus  to 
heat  and  to  desiccation  has  been  supposed  to  be  due  to  the  presence 


BACILLI   IX   CHRONIC    INFECTIOUS   DISEASES.  473 

of  spores.  But,  so  far  as  resistance  to  heat  is  concerned,  this  is  not 
so  great  as  was  at  one  time  believed.  Schill  and  Fischer  (1884),  as- 
suming that  the  tubercle  bacillus  forms  spores,  made  quite  a  number 
of  experiments  to  determine  its  thermal  death-point.  They  sub- 
jected sputum  containing  the  bacillus  to  a  temperature  of  100°  C.,  and 
tested  the  destruction  of  vitality  by  inoculations  into  guinea-pigs. 
Exposure  to  steam  at  a  temperature  of  100°  C.  for  two  to  five  min- 
utes was  effective  in  every  experiment,  with  one  exception.  One 
guinea-pig  died  tuberculous  after  having  been  inoculated  with 
sputum  exposed  to  this  tamperature  for  two  minutes.  This  result 
was  assumed  to  show  that  the  bacillus  would  survive  lower  tempera- 
tures, but  it  is  evident  that  additional  experiments  were  required  to 
establish  this  fact.  In  1887  the  writer  made  a  few  similar  experi- 
ments at  a  lower  temperature,  and  guinea-pigs  inoculated  with  tuber- 
culous sputum  exposed  for  ten  minutes  to  a  temperature  of  90°,  80°, 
and  60°  C.  failed  to  become  tuberculous,  while  another  guinea-pig, 
inoculated  with  the  same  material  after  exposure  to  a  temperature  of 
50°  C.  for  ten  minutes,  died  tuberculous.  These  results  correspond 
with  those  subsequently  (1888)  reported  by  Yersin,  who  tested  the 
thermal  death-point  of  this  bacillus  by  the  culture  method.  This 
author  assumes  that  the  bacilli  form  spores,  but  states  as  a  result  of 
his  experiments  that  "at  the  end  of  ten  days  bacilli  heated  for  ten 
minutes  at  55°  C.  gave  a  culture  in  glycerin-bouillon  ;  those  heated 
to  00°,  at  the  end  of  twenty-two  days;  while  those  heated  to  70°  and 
above  failed  to  grow  in  every  instance.  This  experiment,  repeated  a 
great  number  of  times,  always  gave  the  same  result.  The  tubercle 
bacilli  then  resist  a  temperature  of  60°  C.  for  ten  minutes,  and  it  is 
to  be  remarked  that  the  resistance  of  spores  to  heat  appears  to  be  no 
greater  than  that  of  the  bacilli  themselves."  Yersin  remarks  in  a 
footnote  that  "the  spores  which  served  for  these  experiments  did 
not  appear  as  more  or  less  irregular  granules  taking  the  coloring 
matter  strongly,  but  as  veritable  spores  with  sharply  defined  outlines, 
to  the  number  of  one  or  two  in  a  bacillus,  or  three  at  the  outside. 
These  spores  are  particularly  clear  in  cultures  upon  glycerin-agar 
several  weeks  old." 

It  may  be  that  bacteriologists  have  been  mistaken  in  the  infer- 
ence that  all  spores  possess  a  greater  resisting  power  for  heat  than 
that  exhibited  by  bacilli  in  the  absence  of  spores.  That  this  is  true 
as  regards  anthrax  spores  and  many  others,  the  thermal  death-point 
of  which  has  been  determined  by  exact  experiments,  does  not  prove 
that  it  is  true  for  all.  And  it  is  known  that  there  are  wide  differ- 
ences in  the  resisting  power  both  of  the  spores  of  different  species 
and  in  the  vegetating  cells.  To  admit  that  the  tubercle  bacillus  or 
the  typhoid  bacillus,  etc.,  may  form  spores  which  have  no  greater 


474  BACILLI   IN   CHRONIC    INFECTIOUS   DISEASES. 

resisting  power  against  heat  than  the  bacilli  themselves,  would  there- 
fore simply  be  an  admission  that  soma  bacteriologists  had  made  a 
mistaken  inference  based  upon  incomplete  data.  In  view  of  the 
facts  stated  we  can  simply  repeat  what  was  said  at  the  outset,  viz. , 
the  question  as  to  spore  formation  has  not  been  definitely  deter- 
mined. 

The  tubercle  bacillus  is  a  strict  parasite,  and  its  biological  char- 
acters are  such  that  it  could  scarcely  find  natural  conditions,  outside 
of  the  bodies  of  living  animals,  favorable  for  its  multiplication.  It 
therefore  does  not  grow  as  a  saprophyte  under  ordinary  circum- 
stances. But  it  has  been  noted  by  Roux  and  Nocard  that  when  it 
has  been  cultivated  for  a  time  in  artificial  media  containing  glycerin 
it  may  grow  in  a  plain  bouillon  of  veal  or  chicken,  in  which  media  it 
fails  to  develop  when  introduced  directly  from  a  culture  originating 
from  the  body  of  an  infected  animal.  This  would  indicate  the  pos- 
sibility of  its  acquiring  the  ability  to  grow  as  a  saprophyte  ;  and  we 
can  scarcely  doubt  that  at  some  time  in  the  past  it  was  a  true  sapro- 
phyte. The  experiments  of  Nuttall  indicate  that  the  bacillus  may 
multiply,  under  favorable  temperature  conditions,  in  tuberculous 
sputum  outside  of  the  body.  And  it  is  extremely  probable  that  mul- 
tiplication occurs  in  the  muco-purulent  secretion  which  accumulates 
in  pulmonary  cavities  in  phthisical  patients.  In  these  cavities  its  de- 
velopment may,  in  a  certain  sense,  be  regarded  as  saprophytic,  as  it 
feeds  upon  non-living  organic  material. 

Koch  first  succeeded  in  cultivating  this  bacillus  upon  coagulated 
blood  serum,  prepared  as  directed  in  Section  VIII.,  Part  First,  of  the 
present  volume.  Roux  and  Nocard  have  since  shown  (1888)  that  it 
grows  very  well  on  nutrient  agar  to  which  glycerin  has  been  added 
(six  to  eight  per  cent),  and  also  in  veal  broth  containing  five  per  cent 
of  glycerin.  It  is  difficult  to  obtain  pure  cultures  from  tuberculous 
sputum,  on  account  of  the  presence  of  other  bacteria  which  grow 
much  more  rapidly  and  take  full  possession  of  the  medium  before  the 
tubercle  bacillus  has  had  time  to  form  visible  colonies.  For  this  rea- 
son it  is  best  to  first  inoculate  a  guinea-pig  with  the  tuberculous  spu- 
tum and  to  obtain  cultures  from  it  after  tuberculous  infection  has 
fully  developed.  The  inoculated  animals  usually  die  at  the  end  of 
three  or  four  weeks.  It  is  best  to  kill  one  which  gives  evidence  of 
being  tuberculous,  and  to  remove  one  or  more  nodules  from  the 
lungs  through  an  opening  made  in  the  chest  walls.  The  greatest 
care  will  be  required  to  prevent  contamination  by  other  common 
microorganisms.  The  instruments  used  must  be  sterilized  by  heat, 
and  the  skin  over  the  anterior  thoracic  wall  carefully  turned  back  ; 
then,  after  again  sterilizing  knives  and  scissors,  cut  an  opening  into 
tha  chest  cavity,  draw  out  the  root  of  the  lung,  and  take  up  with 


BACILLI   IX   CHRONIC   INFECTIOUS   DISEASES. 

slender  sterilized  forceps,  or  with  a  strong  platinum  loop,  one  or 
more  well-defined  tubercular  nodules.  These  may  be  conveyed  di- 
rectly to  the  surface  of  the  solid  culture  medium  and  then  broken 
up  and  rubbed  over  the  surface  as  thoroughly  as  possible  ;  or  they 
may  first  be  crushed  between  two  sterilized  glass  slides,  and  then 
transferred  with  the  platinum  loop  and  thoroughly  rubbed  into  the 
surface  of  the  culture  medium. 

This  breaking-up  of  the  tuberculous  nodules  and  distribution  of 
the  bacilli  upon  the  surface  of  the  culture  medium  is  essential  for 
the  success  of  the  experiment.  Instead  of  using  the  tubercular 
nodules  in  the  lungs,  an  enlarged  lymphatic  gland  from  the  axilla  or 
elsewhere  may  be  used,  as  first  recommended  by  Koch.  This  is  to 
be  crushed  in  the  same  way ;  and  it  will  be  best  to  inoculate  a  num- 
ber of  tubes  at  the  same  time,  as  accidental  contamination  or  failure 
to  develop  is  very  liable  to  occur  in  a  certain  number.  Owing  to  the 
liability  of  the  blood  serum  to  become  too  dry  for  the  development  of 
the  bacillus,  it  is  best  to  keep  the  cultures  in  a  moist  atmosphere,  or 
to  prevent  evaporation  by  applying  a  rubber  cap  over  the  open  end 
of  the  test  tube.  This  should  be  sterilized  in  a  solution  of  mercuric 
chloride  (1  : 1,000) ;  and  the  end  of  the  cotton  plug  should  be  burned 
off  just  before  applying  it,  for  the  purpose  of  destroying  the  spores 
of  mould  fungi,  which  in  a  dry  atmosphere  would  be  harmless,  but 
under  the  rubber  cap  are  likely  to  sprout  and  to  send  their  mycelium 
through  the  cotton  plug  to  the  interior  of  the  tube,  thus  destroying 
the  culture. 

Upon  coagulated  blood  serum  the  growth  first  becomes  visible  at 
the  end  of  ten  to  fourteen  days  (at  37°  C.),  and  at  the  end  of  three 
weeks  a  very  distinct  and  characteristic  develop- 
ment has  occurred.    The  first  appearance  is  that  of 
dry-looking,  grayish- white  points  and  scales,  which 
are  without  lustre,  and  are  sometimes  united  to 
form  a  thin,  irregular,  membranous-looking  layer. 
Under  the  microscope,  with  an  amplification  of 
eighty  diameters,  the  early,  thin  surface  growth 
upon  blood  serum  presents  a  characteristic  appear- 
ance.   The  bacilli,  arranged  in  parallel  rows,  form 
variously  curved  figures,  of  which  we  may  obtain 
impressions  by  carefully  applying  a  dry  cover  glass         Fl(r:  f117'~ Tufbe  ^ 
to  the  surface.     Upon  staining  the  preparation  in      culture  upon  blood  se- 
the  usual  way  the  same  arrangement  of  the  bacilli      rum-  x  500-  (Koch-) 
which  adhered  to  the  thin  glass  cover  will  be  pre- 
served.    The    growth  is   more   abundant    in  subsequent   cultures, 
which  have  been  kept  up  in  Koch's  laboratory  from  his  original 
pure  cultures  up  to  the  present  time  ;  in  these  the  bacillus  still   pre- 


476  BACILLI   IN    CHRONIC   INFECTIOUS   DISEASES. 

serves  its  characters  of  form  and  growth,  and  its  specific  pathogenic 
power. 

Pastor  (1892)  has  succeeded  in  obtaining  pure  cultures  of  the 
tubercle  bacillus  from  sputum  by  the  following  ingenious  method  : 
After  proving  by  microscopic  examination  that  the  sputum  of  a 
tuberculous  individual  contains  numerous  bacilli,  he  has  the  patient 
cleanse  his  mouth  as  thoroughly  as  possible  with  sterilized  water, 
and  then  expectorate  some  material,  coughed  up  from  the  lungs,  into 
a  sterilized  test  tube.  By  shaking  with  sterilized  water  a  fine  emul- 
sion is  made,  and  this  is  filtered  through  fine  gauze.  The  filtrate, 
which  is  nearly  transparent,  contains  numerous  tubercle  bacilli.  A 
few  drops  of  the  emulsion  are  now  added  to  liquefied  gelatin  in  a  test 
tube,  and  a  plate  is  made  in  the  usual  way.  This  is  kept  for  three 
or  four  days  at  the  room  temperature,  during  which  time  the  com- 
mon mouth  bacteria  capable  of  growth  form  visible  colonies.  By 
means  of  a  hand  lens  a  place  is  now  selected  in  which  no  colonies  are 
seen,  and  a  bit  of  gelatin  is  excised  with  a  sterilized  knife.  This 
piece  is  transferred  to  the  surface  of  blood  serum  or  glycerin- agar, 
and  placed  in  the  incubating  oven,  where  in  due  time  colonies  of 
the  tubercle  bacillus  will  usually  be  found  to  develop. 

Another  method  of  accomplishing  the  same  result  has  been 
described  by  Kitasato.  This  is  a  method  devised  by  Koch  some  time 
since  and  successfully  employed  in  his  laboratory.  The  morning 
expectoration  of  a  tuberculous  patient,  raised  from  the  lungs  by 
coughing,  is  received  in  a  Petri's  dish.  A  bit  of  sputum,  such  as 
comes  from  the  tuberculous  cavity  in  the  lungs  of  such  a  patient,  is 
now  isolated  with  sterilized  instruments  and  carefully  washed  in  at 
least  ten  successive  portions  of  sterilized  water.  By  this  procedure 
the  bacteria  accidentally  attached  to  the  viscid  mass  of  sputum  dur- 
ing its  passage  through  the  mouth  are  washed  away.  In  the  last 
bath  the  mass  is  torn  apart  and  a  small  portion  from  the  interior  is 
used  to  make  a  microscopic  preparation,  the  examination  of  which 
shows  whether  only  tubercle  bacilli  are  present.  If  this  be  the  case 
cultures  upon  glycerin-agar  are  started  from  material  obtained  from 
the  interior  of  the  same  mass.  The  colonies  obtained  in  this  way 
appear  in  about  two  weeks  as  round,  white,  opaque,  moist,  and  shin- 
ing masses.  Kitasato's  researches  show  that  the  greater  portion  of 
the  tubercle  bacilli  in  sputum  obtained  in  this  way,  and  in  the  con- 
tents of  lung  cavities,  are  incapable  of  development,  although  this 
fact  cannot  be  recognized  by  a  microscopic  examination  of  stained 
specimens. 

On  account  of  the  greater  facility  of  preparing  and  sterilizing 
glycerin-agar,  and  the  more  rapid  and  abundant  development  upon 
this  medium,  it  is  now  usually  employed  in  preference  to  blood 


BACILLI   IN   CHRONIC    INFECTIOUS   DISEASES. 


47: 


Serum.  The  growth  at  the  end  of  fourteen  days  is  more  abundant  than 
upon  blood  serum  at  the  end  of  several  weeks.  When  numerous 
bacilli  have  been  distributed  over  the  surface  of  the  culture  medium 
a  rather  uniform,  thick,  white  layer,  which  subsequently  acquires  a 
yellowish  tint,  is  developed  ;  when  the  bacilli 
are  few  in  number  or  are  associated  in  scattered 
groups  separate  colonies  are  developed,  which 
acquire  considerable  thickness  and  have  more 
or  less  irregular  outlines  ;  they  are  white  at 
first,  then  yellowish-white.  Frankel  describes 
the  tubercle  bacillus  as  a  facultative  anaerobic, 
and  it  would  appear  that  it  must  be  able  to  grow 
in  situations  where  it  can  obtain  very  little  oxy- 
gen from  its  development  in  the  interior  of  tu- 
berculous nodules,  lymphatic  glands,  etc.  But 
in  stick  cultures  in  glycerin-agar  development 
only  occurs  near  the  surface,  and  not  at  all  in 
the  deeper  portion  of  the  medium.  In  view  of 
its  abundant  growth  on  the  surface  it  is  diffi- 
cult to  understand  this  failure-  to  grow  along 
the  line  of  puncture,  if  it  is  in  truth  a  faculta- 
tive anaerobic. 

In  peptonized  veal  broth  containing  five  per 
cent  of  glycerin  the  bacillus  develops  at  first  in 
the  form  of  little  flocculi,  which  accumulate  at 
the  bottom  of  the  flask  and  which  by  agitation 
are  easily  broken  up.  At  the  end  of  two  or 
three  weeks  the  bottom  of  the  flask  is  covered 
with  similar  flocculi,  which  form  an  abundant 
deposit. 

Pawlowski  and  others  report  success  in  cul- 
tivating the  tubercle  bacillus  upon  the  Surface 
of  cooked  potato  enclosed  in  a  test  tube  after 
the  method  of  Bolton  andRoux.  The  open  end 
of  the  tube  is  hermetically  sealed  in  a  flame 
after  the  bacilli  have  been  planted  upon  the 
obliquely-cut  surface  of  the  potato ;  this  prevents  drying.  Ac- 
cording to  Pawlowski,  better  results  are  obtained  if  the  surface  of 
the  potato  is  moistened  with  a  five-per-cent  solution  of  glycerin.  The 
growth  is  said  to  be  seen  at  the  end  of  about  twelve  days  as  grayish, 
dry-looking  flakes  ;  at  the  end  of  three  or  four  weeks  it  forms  a  dry, 
smooth,  whitish  layer,  and  no  further  development  occurs. 

The  range  of   temperature  at  which  this  bacillus  will  grow  is 
very  restricted  ;  37°  C.  is  usually  given  as  the  most  favorable  point, 


FIG.  118.— Culture  of  tu- 
bercle bacillus  upon  glyce- 
rin-agar. Photograph  by 
Roux. 


478  BACILLI  IN  CHRONIC  INFECTIOUS  DISEASES. 

but  Roux  and  RTocard  say  that  the  most  favorable  temperature  ap- 
pears to  be  39°,  and  that  development  is  slower  at  37°. 

The  experiments  of  Koch,  Schill  and  Fischer,  and  others  show 
that  the  bacilli  retain  their  vitality  in  desiccated  sputum  for  several 
months  (nine  to  ten  months — De  Toma) ;  but  they  are  said  to  undergo 
a  gradual  diminution  in  pathogenic  virulence,'  which  is  more  rapid 
when  the  desiccated  material  is  kept  at  a  temperature  of  30°  to  40°  C. 
In  the  experiments  of  Cadeac  and  Malet  portions  of  the  lung  from 
a  tuberculous  cow,  dried  and  pulverized,  produced  tuberculosis  in 
guinea-pigs  at  the  end  of  one  hundred  and  two  days.  They  retain 
their  vitality  for  a  considerable  time  in  putrefying  material  (forty- 
three  days — Schill  and  Fischer  ;  one  hundred  and  twenty  days — Ca- 
deac and  Malet).  The  resisting  power  of  this  bacillus  against  ger- 
micidal  agents  is  also  greater  than  that  of  certain  other  pathogenic 
microorganisms,  but  not  so  great  as  to  justify  the  inference  that  it 
forms  spores.  It  is  not  destroyed  by  the  gastric  juice  in  the  sto- 
mach, as  is  shown  by  successful  infection  experiments  in  suscep- 
tible animals,  by  mixing  cultures  of  the  bacillus  with  their  food 
(Baumgarten,  Fischer),  and  also  by  experiments  with  an  artificially 
prepared  gastric  juice  (Falk).  They  are  destroyed,  in  sputum,  in 
twenty  hours  by  a  three-per-cent  solution  of  carbolic  acid,  even 
when  they  present  the  appearance  usually  ascribed  to  the  presence 
of  spores  (Cavagnis) ;  also  by  absolute  alcohol,  a  saturated  aqueous 
solution  of  salicylic  acid,  saturated  aniline  water,  etc.  (Schill  and 
Fischer).  The  more  recent  experiments  of  Yersin  upon  pure  cul- 
tures of  the  bacillus  gave  the  following  results  :  "  Tubercle  bacilli, 
containing  spores,  were  killed  by  a  five-per-cent  solution  of  carbolic 
acid  in  thirty  seconds,  by  one-per-cent  in  one  minute  ;  absolute  alco- 
hol, five  minutes  ;  iodoform-ether,  one  per  cent,  five  minutes  ;  ether, 
ten  minutes ;  mercuric  chloride,  1  : 1,000  solution,  ten  minutes ; 
thymol,  three  hours  ;  salicylic  acid,  2. 5  per  cent,  six  hours. 

The  tubercle  bacillus  appears  to  be  especially  susceptible  to  the 
action  of  light.  In  his  address  before  the  Tenth  International  Medi- 
cal Congress  (Berlin,  1890)  Koch  says  that  when  exposed  to  direct 
sunlight  the  tubercle  bacillus  is  killed  in  from  a  few  minutes  to  sev- 
eral hours,  according  to  the  thickness  of  the  layer ;  it  is  also  de- 
stroyed by  diffuse  daylight  in  from  five  to  seven  days  when  placed 
near  a  window.  This  fact  has  an  important  hygienic  bearing,  espe- 
cially in  view  of  the  fact  that  the  tubercle  bacillus  is  not  readily 
killed  by  desiccation,  putrefaction  of  the  material  containing  it,  etc. 
Tuberculous  sputum  expectorated  upon  sidewalks,  etc.,  being  ex- 
posed to  the  action  of  direct  sunlight,  will  in  many  cases  be  disin- 
fected by  this  agent  by  the  time  complete  desiccation  has  occurred — 
i.e.,  before  it  is  in  a  condition  to  be  carried  into  the  air  as  dust. 


BACILLI  IN  CHRONIC   INFECTIOUS   DISEASES. 


479 


Sawizky  in  1891  made  a  series  of  experiments  to  determine 
the  length  of  time  during  which  dried  tuberculous  sputum  retains 
its  virulence.  He  arrived  at  the  conclusion  that  virulence  is  not  sud- 
denly but  gradually  lost,  and  that  in  an  ordinary  dwelling  room 
dried  sputum  retains  its  specific  infectious  power  for  two  and  one- 
half  months. 

Tizzoni  and  Cattani  (1892)  have  presented  some  experimental  evi- 
dence which  indicates  that  injections  of  Koch's  tuberculin  into 
guinea-pigs  may  produce  in  these  animals  a  certain  degree  of  im- 
munity against  tuberculosis;  and  that  this  immunity  depends  upon 
the  presence  of  an  anti-tuberculin  formed  in  the  body  of  the  partially 
immune  animal. 

Numerous  experiments  made  by  veterinary  surgeons  upon  tuber- 
culous cows  show  that  the  injection  of  Koch's  tuberculin  in  these 
animals,  in  doses  of  thirty  to  forty  centigrammes,  produces  a  rise  of 
temperature  of  from  1°  to  3°  C.  The  febrile  reaction  usually  occurs 
in  from  twelve  to  fifteen  hours  after  the  injection.  Its  duration  and 
intensity  do  not  depend  upon  the  extent  of  the  tuberculous  lesions, 
but  is  even  more  marked  when  these  are  slight  than  in  advanced 
cases.  In  non-tuberculous  animals  no  reaction  occurs,  and  the  ex- 
periments made  justify  the  suspicion  that  tuberculosis  exists  if  an 
elevation  in  temperature  of  a  degree  or  more  occurs  as  a  result  of 
the  subcutaneous  injection  of  the  dose  mentioned. 

When  the  number  of  tubercle  bacilli  in  sputum  is  comparatively 
small  they  may  easily  escape  observation.  Methods  have  therefore 
been  suggested  for  finding  them  under  these  circumstances.  Ribbert 
(1886)  proposed  the  addition  to  the  sputum  of  a  two-per-cent  solution 
of  caustic  potash,  and  boiling  the  mixture.  The  tenacious  mucus  is 
dissolved,  and  when  the  mixture  is  placed  in  a  conical  glass  vessel 
the  bacilli  are  deposited  at  the  bottom  and  may  easily  be  found  in 
the  sediment  after  removing  the  supernatant  fluid.  The  same  object 
is  accomplished  by  Stroschein  (1889)  by  the  addition  to  sputum  of 
three  times  its  volume  of  a  saturated  solution  of  borax  and  boracic 
acid  in  water. 

A  method  of  estimating  the  number  of  bacilli  in  sputum  has 
been  proposed  by  Nuttall,  which  appears  to  give  sufficiently  ac- 
curate results  and  to  be  useful  in  judging  of  the  progress  of  a 
case  or  of  the  results  of  treatment.  For  the  details  of  this  method 
we  must  refer  to  the  author's  paper  (Johns  Hopkins  Hospital  Bulle- 
tin, vol.  xi.,  No.  13,  1891).  It  consists  essentially  in  first  making 
the  sputum  fluid  by  the  addition  of  a  solution  of  caustic  potash ;  in 
then  shaking  it  thoroughly  in  a  bottle  containing  sterilized  gravel 
or  pounded  glass  ;  in  carefully  measuring  the  total  quantity  of  fluid, 
and  in  dropping  upon  glass  slides  uniform  drops  by  means  of  a  grad- 


480 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


uated  pipette ;  in  spreading  these  uniformly  by  means  of  a  platinum 
needle  and  a  turn  table ;  in  covering  the  dried  film  with  a  film  of 
blood  serum,  and  coagulating  this  by  heat ;  and,  finally,  in  staining 
and  counting  the  bacilli  in  a  series  of  slides  from  the  same  specimen, 
and  from  the  average  number  found  in  a  single  drop  estimating  the 
total  number  in  the  sputum  for  twenty -four  hours. 

Pathogenesis. — Man,  cattle,  and  monkeys  are  most  subject  to 
contract  the  disease  naturally,  and  it  may  be  communicated  by  in- 
oculation to  many  of  the  lower  animals — guinea-pigs,  field  mice,  rab- 


FIG.  119.— Limited  epithelioid  celled  tubercle  of  the  iris.     X  950.    (Bautn^arten  > 

bits,  and  cats  are  among  the  most  susceptible  animals  ;  and  in  larger 
doses  dogs,  rats,  white  mice,  and  fowls  may  also  be  infected. 

When  tuberculous  sputum  is  introduced  beneath  the  skin  of  a 
guinea-pig  the  nearest  lymphatic  glands  are  found  to  be  swollen  at 
the  end  of  two  or  three  weeks,  at  the  same  time  there  is  a  thickening 
of  the  tissues  about  the  point  of  inoculation  ;  later  a  dry  crust  forms 
over  the  local  tuberculous  tumefaction,  and  beneath  this  is  a  flattened 
ulcer  covered  with  cheesy  material.  The  animals  become  emaciated 
and  show  difficulty  in  breathing,  and  usually  succumb  to  general 
tuberculosis,  especially  involving  the  lungs,  within  four  to  eight 
weeks.  Injections  of  tuberculous  sputum,  or  of  pure  cultures  of  the 
bacillus,  into  the  peritoneal  cavity  give  rise  to  extensive  tuberculo- 
sis of  the  liver,  spleen,  and  lungs,  and  to  death,  as  a  rule,  within 
three  or  four  weeks.  Rabbits  are  less  susceptible  to  subcutaneous 


BACILLI   IX   CHRONIC    INFECTIOUS   DISEASES.  481 

injections,  but  die  within  seventeen  to  twenty  clays  when  virulent- 
recent — cultures  are  injected  into  the  circulation.  As  a  result  of 
such  an  inoculation  the  animal :  apidly  loses  flesh  and  has  a  decided 
elevation  of  temperature,  commencing  at  the  end  of  the  first  week 
and  increasing  considerably  during  the  last  days  of  life.  At  the 
autopsy  the  spleen  and  liver  are  found  to  be  greatly  enlarged,  but 
they  do  not  contain  any  tubercles  that  can  be  recognized  by  the  naked 
eye  (Yersin).  They  contain,  however,  great  numbers  of  tubercle 
bacilli,  both  free  and  in  the  cells.  Injections  of  a  small  quantity  of 
a  pure  culture  into  the  anterior  chamber  of  the  rabbit's  eye  cause 
first  iris-tuberculosis,  f ollowed  by  swelling  and  caseation  of  the  near- 
est lymph  glands,  and  finally  general  infection  and  death  ;  when 
larger  quantities  are  injected  general  tuberculosis  is  quickly  devel- 
oped. The  influence  of  quantity — number  of  bacilli — is  also  shown 
in  subcutaneous,  intravenous,  or  intraperitoneal  injections  into  guinea- 
pigs  and  rabbits  (Hirschberger,  G-ebhardt,  Wyssokowitsch).  Thus 
rabbits  which  received  less  than  one  hundred  and  fifty  bacilli,  in 
sputum,  in  the  experiments  of  Wyssokowitsch,  did  not  develop  tuber- 
culosis ;  and  in  guinea-pigs  the  smaller  the  number  injected  the  more 
protracted  the  course  of  the  disease  was  found  to  be. 

Tuberculosis  in  man  no  doubt  results,  in  a  large  proportion  of  the 
cases,  from  the  respiration,  by  a  susceptible  individual,  of  air  con- 
taining the  tubercle  bacillus  in  suspension  in  a  desiccated  condition. 
As  already  stated,  it  has  been  demonstrated  by  experiment  that  the 
bacillus  retains  its  vitality  in  desiccated  sputum  for  several  months. 
The  experiments  of  Cornet  have  demonstrated  that  in  the  dust  of 
apartments  occupied  by  tuberculous  patients  tubercle  bacilli  are  very 
commonly  present  in  sufficient  numbers  to  induce  tuberculosis  in 
guinea-pigs  inoculated  in  the  peritoneal  cavity  with  such  dust,  while 
negative  results  were  obtained  from  inoculations  with  dust  from 
other  localities.  In  view  of  these  facts  the  usual  mode  of  infection 
is  apparent.  Infection  may  also  occur  through  an  open  wound  or 
abrasion  of  the  skin,  as  in  the  small,  circumscribed  tumors  which 
sometimes  develop  upon  the  hands  of  pathologists  as  a  result  of 
handling  tuberculous  tissues.  A  few  instances  of  accidental  inocu- 
lation through  wounds  made  by  glass  or  earthen  vessels  containing 
tuberculous  sputum  have  also  been  recorded.  A  more  common  mode 
of  infection,  especially  in  children,  is  probably  by  way  of  the  intesti- 
nal glands,  from  the  ingestion  of  the  milk  of  tuberculous  cows.  That 
infection  may  occur  by  way  of  the  intestine  has  been  proved  by  ex- 
periments upon  rabbits,  which  develop  tuberculosis  when  fed  upon 
tuberculous  sputum.  And  that  the  tubercle  bacillus  is  frequently,  if 
not  usually,  present  in  the  milk  of  tuberculous  cows  has  been  proved 
by  the  experiments  of  Bellinger,  Hirschberger,  Ernst,  and  others. 
31 


482  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

In  Hirschberger's  investigations  milk  from  tuberculous  cows  in- 
duced tuberculosis  in  guinea-pigs,  when  .injected  subcutaneously  or 
into  the  peritoneal  cavity,  in  fifty-five  per  cent  of  the  cases  studied 
(twenty).  The  conclusion  is  reached  that  the  milk  may  contain  tu- 
bercle bacilli  even  when  the  udder  of  the  cow  is  not  involved.  Ernst 
also,  from  an  examination  of  the  milk  from  thirty-six  tuberculous 
cows  in  which  the  udder  was  apparently  not  involved,  found  the 
tubercle  bacillus  by  microscopical  examination  in  five  per  cent  of  the 
samples  examined  (one  hundred  and  fourteen). 

The  prevalence  of  tuberculosis  among  cattle  is  shown  by  numer- 
ous investigations,  and  especially  by  the  official  inspections  of 
slaughtered  animals  made  in  Germany.  Thus  in  Saxony,  in  the 
year  1889,  of  611,511  cattle  examined  6,135  were  found  to  be  tubercu- 
lous (about  one  per  cent) ;  in  Berlin,  1887-1888,  out  of  130,733  ani- 
mals slaughtered  4,300  were  found  to  be  tuberculous  (3.2  per  cent). 
In  view  of  the  facts  stated  the  great  mortality  from  tubercular  dis- 
eases among  children,  many  of  whom  are  removed  from  other  prob- 
able sources  of  infection,  is  not  difficult  to  understand,  and  the 
practical  and  simple  method  of  preventing  infection  in  this  way,  af- 
forded by  the  sterilization  (by  heat)  of  milk  used  as  food  for  infants, 
must  commend  itself  to  all. 

BACILLUS   TUBERCULOSIS   GALLINARUM. 

The  researches  of  Maffucci  (1889)  and  of  Cadiot,  Gilbert,  and 
Roger  (1890)  show  that  the  bacillus  obtained  from  spontaneous  tu- 
berculosis in  chickens,  although  closely  resembling  the  bacillus  of 
human  tuberculosis,  is  not  identical  with  it,  varying  especially  in  its 
pathogenic  power.  This  view  is  sustained  by  the  observations  of 
Koch,  who  says  in  his  address  before  the  Tenth  International  Medi- 
cal Congress  (Berlin,  1890) : 

"The  care  which  it  is  necessary  to  exercise  in  judging  of  the  characters 
which  serve  to  differentiate  bacteria,  even  those  which  are  well  known,  I 
have  learned  in  the  case  of  the  tubercle  bacillus.  This  species  is  so  definitely 
characterized  by  its  staining  reactions,  its  growth  in  pure  cultures,  and  its 
pathogenic  qualities,  and  indeed  by  each  of  these  characters,  that  it  seems 
impossible  to  confound  it  with  other  species.  Nevertheless  in  this  case  also 
one  should  not  rely  upon  a  single  one  of  the  characters  mentioned  for  de- 
termining the  species,  but  should  follow  the  safe  rule  that  all  available 
characters  should  be  considered,  and  the  identity  of  a  certain  bacterium 
should  only  be  regarded  as  demonstrated  when  it  has  been  shown  to  corre- 
spond in  all  of  these  particulars  When  I  made  my  first  researches  with 
reference  to  the  tubercle  bacillus  I  was  controlled  by  this  rule,  and  tested 
tubercle  bacilli  from  various  sources,  not  only  with  reference  to  their  stain- 
ing reactions,  but  also  with  reference  to  their  growth  in  culture  media  and 
pathogenic  characters.  Only  in  the  tuberculosis  of  chickens  I  was  not  able 
to  apply  this  rule,  as  at  that  time  it  was  not  possible  for  me  to  obtain  fresh 
material  from  which  to  make  pure  cultures.  As,  however,  all  other  forms 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  483 

of  tuberculosis  had  given  identical  bacilli,  and  the  bacilli  of  chicken  tuber- 
culosis in  their  appearance  and  behavior  towards  the  aniline  colors  entirely 
corresponded  with  these,  I  believed  myself  justified  in  assuming  their  iden- 
tity, notwithstanding  the  incompleteness  of  the  research.  Later  I  received 
pure  cultures  from  various  sources,  which  apparently  originated  from  tuber- 
cle bacilli,  but  in  several  regards  differed  from  these ;  especially  in  the  fact 
that  inoculation  experiments,  made  by  experienced  and  reliable  investigators, 
led  to  dissimilar  results,  which  it  was  necessary  to  regard  as  unexplained  con- 
tradictions. At  first  I  believed  that  these  differences  depended  upon  changes 
such  as  are  frequently  observed  in  pathogenic  bacteria,  when  these  are  culti- 
vated in  pure  cultures  outside  of  the  body  for  a  long  time  under  more  or  less 
unfavorable  conditions.  In  order  to  solve  the  riddle  I  attempted  by  various 
influences  to  change  the  common  tubercle  bacilli  into  the  presumed  variety 
referred  to.  They  were  cultivated  for  several  months  at  so  high  a  tempera- 
ture that  only  a  scanty  growth  was  obtained;  in  other  experiments  still 
higher  temperatures  were  allowed  to  act  repeatedly  for  so  long  a  time  that 
the  cultures  were  brought  as  nearly  as  possible  to  the  point  of  killing  the 
bacilli.  In  a  similar  way  I  subjected  the  cultures  to  the  action  of  chemical 
agents,  of  light,  or  absence  of  moisture ;  they  were  cultivated  for  many  gen- 
erations in  association  with  other  bacteria ;  inoculated  successively  in  ani- 
mals having  but  a  slight  susceptibility.  But,  in  spite  of  all  these  attempts, 
only  slight  variations  were  obtained  in  their  characters — far  less  than  other 
pathogenic  bacteria  undergo  under  similar  circumstances.  Itappears,  there- 
fore, that  the  tubercle  bacilli  retain  their  characters  with  special  obstinacy ; 
this  is  in  accord  with  the  fact  that  pure  cultures  which  have  now  been  cul- 
tivated by  me  in  test  tubes  for  more  than  nine  years,  without  in  the  mean- 
time having  been  in  a  living  body,  are  still  entirely  unchanged  with  the  ex- 
ception of  a  slight  diminution  of  virulence.  ...  It  happened  about  a  year 
ago  that  I  received  a  living  chicken  which  was  suffering  from  tuberculosis, 
and  I  used  this  opportunity  to  make  cultures  directly  from  the  diseased  or- 
gans of  this  animal,  which  previously  I  had  not  been  able  to  do.  When  the 
cultures  grew  I  saw  to  my  surprise  that  they  had  precisely  the  appearance 
and  all  of  the  characters  possessed  by  the  enigmatical  cultures  resembling 
those  of  the  genuine  tubercle  bacillus.  Later  I  learned  that  these  also  ori- 
ginated from  tuberculosis  in  fowls,  but,  upon  the  assumption  that  all  forms 
of  tuberculosis  are  identical,  had  been  considered  genuine  tubercle  bacilli. 
A  verification  of  my  observations  I  find  in  the  recently  published  researches 
of  Prof.  Maffucci  with  reference  to  tuberculosis  of  fowls." 

According  to  Maffucci,  adult  chickens  are  refractory  against  the 
action  of  the  Bacillus  tuberculosis  from  man,  and  there  are  slight 
morphological  and  biological  differences  in  the  bacilli  from  the  two 
sources. 

Cadiot,  Gilbert,  and  Roger  (1891)  have  made  a  series  of  experi- 
ments with  the  bacillus  of  tuberculosis  in  fowls.  They  found 
the  bacilli  to  be  very  numerous  in  the  livers  of  chickens  suffering 
from  spontaneous  tuberculosis,  and  inoculated  with  material  from 
this  source  six  chickens,  five  rabbits,  and  twelve  guinea-pigs.  The 
chickens,  when  inoculated  in  the  cavity  of  the  abdomen  or  by  injec- 
tion into  a  vein,  died  in  from  forty-one  to  ninety-three  days  from 
general  tuberculosis.  Four  of  the  rabbits  died  of  general  tuberculosis, 
presenting  the  same  appearance  as  that  following  inoculation  with 
bacilli  from  human  tuberculosis.  Of  the  guinea-pigs,  which  were 
inoculated  in  the  cavity  of  the  abdomen,  eleven  remained  in  good 


484  BACILLI  IN   CHRONIC   INFECTIOUS   DISEASES. 

health  and  one  only  died  of  general  tuberculosis.  These  experi- 
ments show  a  decided  difference  in  the  pathogenic  properties  of 
tubercle  bacilli  from  the  two  sources,  for  the  guinea-pig  is  especially 
susceptible  to  tuberculosis  as  a  result  of  similar  inoculations  with 
bacilli  from  human  tuberculosis.  We  must  therefore  conclude  that 
the  bacillus  found  in  spontaneous  tuberculosis  in  fowls  is  a  distinct 
variety  of  Bacillus  tuberculosis.  Whether  this  variety  would  cause 
tuberculosis  in  man,  if  introduced  into  susceptible  subjects,  has  not 
been  determined ;  and,  as  pointed  out  by  Koch,  this  question  can 
only  be  answered  in  the  affirmative  if  it  should  be  obtained  in  pure 
cultures  from  cases  of  human  tuberculosis. 

Since  the  above  was  written  Maffucci  has  published  (1892)  an 
elaborate  memoir  upon  tuberculosis  of  fowls.  His  conclusions  are 
stated  as  follows : 

"  The  bacillus  of  tuberculosis  in  fowls  is  distinguished  from  that  of  tuber- 
culosis in  mammals  by  the  following  points  of  difference: 

"1.  It  does  not  induce  tuberculosis  in  guinea-pig's,  and  seldom  causes 
general  tuberculosis  in  rabbits. 

' '  2.  The  cultures  in  various  media  have  a  different  appearance  from  those 
of  the  Bacillus  tuberculosis  of  mammals. 

"  3.  The  temperature  at  which  it  develops  varies  between  35°  and  45°  C., 
and  the  thermal  death-point  is  70°  C. 

"4.  At  45°  to  50°  C.  the  cultures  show  long,  thick,  and  branched  forms. 

"5.  The  bacillus  retains  its  vegetative  and  pathogenic  power  at  the  end 
of  two  years. 

"  6.  This  bacillus  produces  a  substance  which  is  toxic  for  guinea-pigs  and 
is  but  slightly  toxic  for  grown  fowls. 

"  7.  The  tuberculosis  produced  in  fowls  by  this  bacillus  is  without  giant 
cells." 

Additional  Notes  upon  the  Tubercle  Bacillus  (1895).— Several 
authors  (Metschnikoff,  Czaplewski,  Fischel)  have  described  branch- 
ing forms  of  the  tubercle  bacillus,  and  Lubinsky  (1895)  reports  that 
in  certain  media  it  grows  out  into  long  threads,  which,  however,  he 
has  never  observed  to  be  branched.  The  media  used  by  him  are  said 
to  give  a  more  abundant  growth  than  occurs  upon  glycerin-agar ; 
the  most  favorable  being  made  of  flesh-peptone  agar,  or  flesh-peptone 
bouillon,  containing  four  per  cent  of  glycerin  and  mashed  potato, 
one  kilo  of  finely  chopped  and  washed  potato  to  fifteen  hundred  cubic 
centimetres  of  water;  this  is  cooked  for  three  or  four  hours  and  filtered 
— to  the  filtrate  is  added  four  per  cent  of  glycerin ;  one  and  a  half 
per  cent  of  agar  is  now  added  and  the  mixture  is  again  cooked  and 
filtered. 

Jones  (1895)  has  observed  the  branching  forms  previously  de- 
scribed by  several  authors,  and  states  that  they  are  only  found  upon 
the  surface  of  culture  media  where  there  is  free  access  of  oxygen. 
He  concludes  that  the  tubercle  bacillus  does  not  form  endogenous 


BACILLI  IN  CHRONIC   INFECTIOUS   DISEASES.  485 

spores,  such  as  are  found  in  various  other  bacilli,  but  that  in  the  rods 
and  branched  filaments  certain  objects  are  seen  which  are  probably 
reproductive  elements,  and  which  closely  resemble  similar  bodies 
("  Kolben  ")  seen  in  the  actinomyces  fungus,  to  which  Jones  believes 
the  tubercle  bacillus  is  closely  related. 

Prudden  and  Hodenpyl  (1891)  have  shown  that  the  injection  of 
dead  tubercle  bacilli  in  rabbits  gives  rise  to  the  development  of  nod- 
ules in  the  lung  containing  epithelioid  and  giant  cells,  but  that  these 
never  undergo  caseation.  This  fact  is  supposed  to  justify  the  infer- 
ence that  caseation  is  due  to  the  products  elaborated  during  the 
growth  of  living  tubercle  bacilli.  The  results  reported  by  Vissmann 
(1892)  correspond  with  those  reported  by  Prudden  and  Hodenpyl. 
Gamaleia  (1892)  has  also  obtained  nodules  with  epithelioid  and 
giant  cells  from  the  injection  of  dead  tubercle  bacilli,  but  in  his  ex- 
periments he  also  found  caseation  of  the  nodules.  Baumgarten  sug- 
gests that  this  was  probably  due  to  the  fact  that  there  were  some  liv- 
ing tubercle  bacilli  remaining  in  the  cultures  which  he  injected. 

Loomis  (1890)  and  Pizzini  (1892)  have  shown  that  living  tubercle 
bacilli  are  not  infrequently  found  in  the  bronchial  glands  of  individ- 
uals who  present  no  evidence  of  tubercular  disease  of  the  lungs  or  else- 
where. The  author  last  mentioned  inoculated  thirty  guinea-pigs 
with  the  bronchial,  mesenteric,  and  cervical  glands  of  thirty  in- 
dividuals in  whom  death  was  due  to  accident  or  acute  disease,  and, 
who  were  free  from  tuberculosis.  Twelve  of  these  thirty  guinea- 
pigs  developed  tuberculosis  as  a  result  of  the  inoculation. 

Straus  (1894)  has  found  tubercle  bacilli  in  the  nasal  cavities  of 
healthy  individuals. 

Ernst  (1895),  as  the  result  of  extended  researches  made  under  the 
auspices  of  the  Massachusetts  Society  for  Promoting  Agriculture, 
has  arrived  at  the  following  conclusions  with  reference  to  the  pres- 
ence of  the  tubercle  bacillus  in  the  milk  of  tuberculous  cows : 

"  The  possibility  of  milk  from  tuberculous  udders  containing  the 
infectious  element  is  undeniable. 

"  With  the  evidence  here  presented,  it  is  equally  undeniable  that 
milk  from  diseased  cows  with  no  appreciable  lesion  of  the  udder  may, 
and  not  infrequently  does,  contain  the  bacillus  of  the  disease." 

De  Schweinitz  (1894)  has  found  that  by  continued  cultivation  in 
an  artificial  medium  the  tubercle  bacillus  becomes  attenuated,  so  that 
when  inoculated  into  guinea-pigs  these  animals  give  no  evidence  of 
tubercular  infection  for  six  months  or  more.  And  his  experiments 
indicate  that  animals  which  have  survived  an  inoculation  with  the 
attenuated  tubercle  bacillus  acquire  an  immunity  against  the  patho- 
genic action  of  virulent  cultures. 


486  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

Amann  (1895)  has  given  in  the  Centralblatt  fur  Bakteriologie 
(Bd.  xvii.,  page  513)  a  detailed  account  of  his  method  for  demon- 
strating the  presence  of  tubercle  bacilli  in  sputum  by  sedimentation. 
He  mixes  the  sputum  with  two  to  four  volumes  of  cold  distilled 
water,  in  a  glass  cylinder  which  should  not  be  more  than  half  full. 
He  adds  one  cubic  centimetre  of  chloroform  and  a  small  quantity 
of  shot;  the  glass  cylinder  is  then  closed  with  a  rubber  cork  and  vio- 
lently shaken  for  some  minutes.  From  four  to  six  volumes  of  dis- 


^^i^  '"••:  I  ^ 

FIG.  120.  —Section  of  a  recent  lepra  nodule  of  the  skin,    x  950.    (Baumgarten.) 

tilled  water  are  then  added  and  the  mixture  is  placed  in  a  V-formed 
glass  tube  for  sedimentation ;  two  cubic  centimetres  of  carbol-fuchsin 
solution  are  added  and  distributed  by  gentle  agitation  of  the  tube. 
At  the  end  of  two  days  the  sedimentation  is  complete  and  the  stained 
bacilli,  cells,  connective-tissue  fibres,  etc.,  are  taken  up  with  a  pipette 
for  examination  under  the  microscope. 

BACILLUS   LEPRJE. 

Discovered  by  Hansen  (1879),  chiefly  in  the  interior  of  the  peculiar 
round  or  oval  cells  found  in  leprous  tubercles.  Discovery  confirmed 
by  Neisser  (1879)  and  by  many  subsequent  observers. 

While  found  chiefly  in  the  leprous  tubercles  of  the  skin  and  mucous 
membranes,  the  bacilli  have  also  been  found  in  the  lymphatic  glands, 
the  liver,  the  spleen,  the  testicles,  and,  in  the  anaesthetic  form  of  the 
disease,  in  the  thickened  portions  of  nerves  involved  in  the  leprous 
process.  Some  observers  have  also  reported  finding  them  in  the 
blood,  but  this  appears  to  be  quite  exceptional.  In  the  leprous  cells 
they  are  commonly  found  in  great  numbers,  and  they  may  also  be 
seen  in  the  lymph  spaces  outside  of  these  cells.  They  are  not  found 
in  the  epidermal  layer  of  the  skin,  but,  according  to  Babes,  they  may 
penetrate  the  hair  follicles. 

Morphology. — The  bacillus  of  leprosy  resembles  the  tubercle  ba- 
cillus in  form,  but  is  of  more  uniform  length  and  not  so  frequently 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  487 

bent  or  curved.  The  rods  have  pointed  ends  ;  and  in  stained  pre- 
parations unstained  spaces,  similar  to  those  observed  in  the  tubercle 
bacillus  and  generally  assumed  to  be  spores,  are  to  be  seen,  although 
not  quite  so  distinctly  as  in  the  latter.  The  bacilli  are  said  by  Fliigge 
to  be  from  four  to  six  ^  in  length  and  less  than  one  /<  in  width — 
probably  considerably  less,  for  the  same  author  states  that  the  tubercle 
bacillus  has  about  the  diameter  of  the  bacillus  of  mouse  septicaemia, 
and  this  is  given  as  0.2  />.. 

This  bacillus  stains  readily  with  the  aniline  colors  and  also 
by  Gram's  method.  Although  it  differs  from  the  tubercle  bacillus 
in  the  ease  with  which  it  takes  up  the  ordinary  aniline  colors,  it  re- 
sembles it  in  retaining  its  color  when  subsequently  treated  with 
strong  solutions  of  the  mineral  acids.  Double-stained  prepara- 
tions are  therefore  easily  made  by  first  staining  sections  or  cover- 
glass  preparations  in  Ziehl's  carbol-f  uchsiii  solution  or  in  an  aqueous 
solution  of  methyl  violet,  decolorizing  in  acid,  washing  in  alcohol, 
and  counter-staining  with  methylene  blue — or,  if  methyl  violet  was 
used  in  the  first  instance,  with  vesuvin. 

Biological  Characters. — The  earlier  attempts  to  cultivate  this 
bacillus  were  without  success,  but  recently  Bordoni-Uffreduzzi  has 
obtained  from  the  marrow  of  the  bones  of  a  leper  a  bacillus  which 
he  believes  to  be  the  leprosy  bacillus,  and  which  he  was  able  to  culti- 
vate upon  blood  serum  to  which  a  certain  amount  of  peptone  and  of 
glycerin  had  been  added.  At  first  this  bacillus  only  grew  with  diffi- 
culty and  in  the  incubating  oven  ;  but  after  it  had  been  cultivated 
artificially  through  a  number  of  generations  it  is  said  to  have  grown 
upon  ordinary  nutrient  gelatin  at  the  room  temperature.  The  bacillus 
obtained  in  this  way  is  said  to  have  retained  its  color  when  treated 
with  acids,  after  having  been  stained  with  aniline-f uchsin,  correspond- 
ing in  this  respect  with  the  bacillus  of  leprosy  and  the  tubercle  ba- 
cillus. But  it  differed  considerably  in  its  morphology  from  the  Ba- 
cillus leprse  as  seen  in  the  tissues  of  lepers,  being  considerably  thicker, 
and  it  was  not  so  promptly  stained  by  the  aniline  colors  as  is  the 
bacillus  found  in  the  tissues.  Moreover,  attempts  to  cultivate  the 
same  bacillus  from  leprous  tubercles  of  the  skin  were  unsuccessful, 
as  were  also  inoculation  experiments  into  the  anterior  chamber  of  the 
eye  in  rabbits.  It  is  therefore  a  matter  of  doubt  as  to  whether  the 
bacillus  obtained  by  Bordoni-Uffreduzzi  is  identical  with  that  present 
in  such  numbers  in  the  cells  of  the  leprous  tubercles,  to  which  the 
name  Bacillus  lepra?  has  been  given. 

Some  of  the  earlier  observers  described  the  bacillus  of  leprosy  as 
motile,  but  this  assertion  seems  to  have  been  based  upon  some  error 
of  observation,  and  it  is  now  generally  agreed  that,  like  the  tubercle 
bacillus,  it  is  without  proper  movements.  The  question  of  spore  for- 


488  BACILLI   IN    CHRONIC   INFECTIOUS   DISEASES. 

mation  has  not  been  definitely  settled.  As  before  remarked,  un- 
stained portions,  occurring  at  regular  intervals,  are  seen  in  the  rods  in 
stained  preparations  ;  but  no  satisfactory  evidence  has  been  presented 
to  show  that  these  are  truly  reproductive  spores. 

Pathogenesis. — The  inference  that  the  bacillus  above  described 
bears  an  etiological  relation  to  the  disease  with  which  it  is  associated 
is  based  upon  the  demonstration  of  its  constant  presence  in  leprous 
tissues — which  has  now  been  repeatedly  made  in  various  and  distant 
parts  of  the  world — and  of  its  absence  from  the  same  tissues  involved 
in  different  morbid  processes.  As  it  has  not  been  obtained  in  pure 
cultures,  the  final  proof  of  such  etiological  relation  is  still  wanting. 
We  have,  however,  experimental  evidence  to  show  that  leprous  tis- 
sues containing  this  bacillus  are  infectious  and  may  reproduce  the 
disease.  The  experiment  has  been  made  upon  man  by  Arning,  who 
inoculated  a  condemned  criminal  subcutaneously  with  fresh  leprous 
tubercles.  The  experiment  was  made  in  the  Sandwich  Islands,  and 
the  man  was  under  observation  until  his  death  occurred  from  leprosy 
at  the  end  of  about  five  years.  The  first  manifestations  of  the  disease 
became  visible  in  the  vicinity  of  the  point  of  inoculation  several 
months  after  the  experimental  introduction  of  the  infectious  material. 

Positive  results  have  also  been  reported  in  the  lower  animals  by 
Damsch,  by  Vossius,  and  by  Melcher  and  Ortmann.  The  last-named 
investigators  inoculated  rabbits  in  the  anterior  chamber  of  the  eye 
with  portions  of  leprous  tubercles  excised  for  the  purpose  from  a 
leper.  The  animals  died  from  general  infection  at  the  end  of  several 
months,  and  the  characteristic  tubercles  containing  the  bacillus  were 
distributed  through  the  various  organs. 

Wolters  (1893)  who  has  made  numerous  inoculation  experiments 
and  has  made  a  critical  review  of  all  the  recorded  experimental  evi- 
dence, arrives  at  the  conclusion  that  the  comparatively  small  number 
of  successful  results  reported  cannot  be  accepted  as  evidence  that 
leprosy  can  be  transmitted  to  the  lower  animals  by  inoculation.  He 
believes  that  in  some  cases  the  tubercle  bacillus  has  been  present  in 
the  material  inoculated  and  that  the  infectious  process  following  the 
inoculation  was  tuberculous  and  not  leprous.  In  inoculations  into 
the  anterior  chamber,  in  the  eyes  of  rabbits,  the  considerable  number 
of  bacilli  introduced  with  the  leprous  tissue  remain  and  retain  their 
staining  properties,  so  that  the  bacilli  originally  introduced  are  found 
in  the  leucocytes  of  the  inflammatory  exudate  or  granulation  tissue 
formed  as  a  result  of  the  introduction  of  foreign  material.  Wolters 
also  doubts  whether  the  few  successful  results  reported  in  the  culti- 
vation of  the  lepra  bacillus  are  trustworthy.  He  has  never  succeeded 
in  his  efforts  to  cultivate  the  bacillus. 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


489 


BACILLUS   MALLEI. 

Synonyms. — The  bacillus  of  glanders;  Der  Rotzbacillus,  Ger\; 
Bacille  de  la  morve,  Fr. 

Discovered  by  Loffler  and  Schiitz  (1882),  and  proved  to  be  the 
cause  of  glanders  by  the  successful  inoculation  of  pure  cultures. 
Found  especially  in  the  recent  nodules  in  animals  infected  with 
glanders  ;  also  in  the  same  after  ulceration,  and  in  the  discharge 
from  the  nostrils,  pus  from  the  specific  ulcers,  etc. ;  sometimes  in  the 
blood  of  infected  animals  (Weichselbaum). 

Morphology. — Bacilli  with  rounded  ends,  straight  or  slightly 
curved,  rather  shorter  and  decidedly  thicker  than  the  tubercle  bacil- 
lus ;  usually  solitary,  but  occasionally  united  in 
pairs,  or  in  filaments  containing  several  elements 
(in  potato  cultures).  In  stained  preparations 
unstained  or  feebly  stained  spaces  are  seen  in 
the  rods,  alternating  with  the  deeply  stained 
protoplasm  of  the  cell.  As  in  the  tubercle  bacil- 
lus, which  presents  a  similar  appearance,  these 
spaces-  have  been  supposed  by  some  bacteriolo- 
gists to  represent  spores  ;  but  Loffler  believes 
them  to  represent  rather  a  degeneration  of  the 
protoplasm.  Baumgarten  and  Rosenthal  claim 
to  have  demonstrated  the  presence  of  spores  by  the  use  of  Weisser's 
method  of  staining,  but  they  do  not  consider  it  established  that  the 
unstained  spaces  in  the  rods  referred  to  are  of  this  nature. 

The  glanders  bacillus  may  be  stained  with  aqueous  solutions  of 
the  aniline  colors,  but  the  staining  is  more  intense  when  the  solution 


V 


FIG.  121.— Bacillus  mal- 
lei, x  1,000.  From  a  pho- 
tomicrograph. CFninkel 
and  Pfeiffer.) 


ptre 

-3<j*y-!fl5 
FIG.  122.— Section  of  a  glanders  nodule,    x  700.    (Fliigge.) 

is  made  feebly  alkaline.  Add  to  three  cubic  centimetres  of  a  1: 10,000 
solution  of  caustic  potash,  in  a  watch  glass,  one  cubic  centimetre  of 
a  saturated  alcoholic  solution  of  an  aniline  color  (methylene  blue, 


490  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

gentian  violet  or  fuchsin) ;  or  the  aniline- water-fuchsin,  or  methyl 
violet  solution  of  Ehrlich  may  be  used,  with  the  addition  just  be- 
fore use  of  an  equal  quantity  of  1  : 10,000  solution  of  caustic  potash. 
Loffler  recommends  that  cover-glass  preparations  be  placed  in  Ehr- 
lich's  solution  and  heated  for  five  minutes;  then  decolorized  in  a  one- 
per-cent  solution  of  acetic  acid  to  which  sufficient  tropseolin  has 
been  added  to  give  it  the  yellow  color  of  Rhine  wine  ;  then  quickly 
washed  in  distilled  water.  This  bacillus  presents  the  peculiarity  of 
losing  very  quickly  in  decolorizing  solutions  the  color  imparted  to  it 
by  the  aniline  staining  solutions.  For  this  reason  the  staining  of  the 
bacillus  in  sections  is  attended  with  some  difficulty.  Loffler  recom- 
mends his  alkaline  methylene-blue  solution  for  staining  sections  ;  and 
for  decolorizing,  a  mixture  containing  ten  cubic  centimetres  of  distilled 
water,  two  drops  of  strong  sulphuric  acid,  and  one  drop  of  a  five- 
per-cent  solution  of  oxalic  acid.  Thin  sections  should  be  left  in  this 
acid  solution  about  five  seconds.  The  method  more  recently  recom- 
mended by  Kuhne  also  gives  good  results  in  skilful  hands  (see  p.  35). 
Biological  Characters. — An  aerobic,  non-motile,  parasitic 
bacillus,  which  may  be  cultivated  in  various  artificial  media  at  a 
temperature  of  37°  C.  The  lowest  temperature  at  which  develop- 
ment occurs  (22°  C. — Loffler)  is  a  little  above  that  at  which  nutrient 
gelatin  is  liquefied  ;  the  highest  limit  is  43°  C.  According  to  Frankel, 
the  glanders  bacillus  is  a  facultative  anaerobic.  Baumgarten  and 
Rosenthal  claim  to  have  demonstrated  the  presence  of  spores  by 
Neisser's  method  of  staining.  Loffler  was  led  to  doubt  the  forma- 
tion of  spores  from  the  results  of  his  experiments  upon  the  thermal 
death-point  of  this  bacillus,  and  its  comparatively  slight  resistance 
to  desiccation  and  destructive  chemical  agents.  He  found  that  ex- 
posure for  ten  minutes  to  a  temperature  of  55°  C.,  or  for  five  minutes 
to  a  three-  to  five-per-cent  solution  of  carbolic  acid,  or  for  two  min- 
utes to  a  1  :  5,000  solution  of  mercuric  chloride,  was  effectual  in  de- 
stroying its  vitality.  As  a  rule,  the  bacilli  do  not  grow  after  having 
been  preserved  in  a  desiccated  condition  for  a  few  weeks  ;  and  in  a 
moist  condition  the  cultures  cannot  be  preserved  longer  than  three 
or  four  months — usually  not  so  long  as  this  (Loffler).  The  bacillus 
does  not  grow  in  infusions  of  hay,  straw,  or  horse  manure,  and  it  is 
doubtful  whether  it  finds  conditions  in  nature  favorable  for  its  sap- 
rophytic  existence.  It  grows,  in  the  incubating  oven,  in  neutral 
bouillon,  in  nutrient  gelatin,  or  in  nutrient  agar,  and  still  better  in 
glycerin-agar.  Upon  the  last-mentioned  medium  it  grows,  even  at 
the  room  temperature  (Kranzfeld),  but  better  still  in  the  incubating 
oven,  as  a  pale- white,  transparent  streak  along  the  line  of  inocula- 
tion, which  at  the  end  of  six  or  seven  days  may  have  a  width  of 
seven  to  eight  millimetres.  According  to  Raskina,  nutrient  agar 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  491 

made  with  milk  forms  an  extremely  favorable  medium,  upon  which 
a  thick,  pale-white  layer  develops  in  two  or  three  days,  which  on  the 
third  or  fourth  day  acquires  an  amber-yellow  color,  and  the  deeper 
layers  acquire  a  brownish-red  tint. 

The  growth  upon  solidified  blood  serum,  in  the  course  of  three  or 
four  days  at  37°  C.,  consists  of  yellowish,  transparent  drops,  which 
later  coalesce  into  a  viscid  layer,  which  has  a  milky  appearance  from 
the  presence  of  numerous  small  crystals  (Baumgarten).  The  growth 
upon  cooked  potato  is  especially  characteristic.  In  the  incubating 
oven,  at  the  end  of  two  or  three  days,  a  rather  thin,  yellowish,  trans- 
parent layer  develops,  which  resembles  a  thin  layer  of  honey.  Later 
this  ceases  to  be  transparent,  and  the  amber  color  changes,  at  the 
end  of  six  to  eight  days,  to  a  reddish-brown  color ;  and  outside  of 
the  reddish-brown  layer,  with  more  or  less  irregular  outlines,  the 
potato  for  a  short  distance  acquires  a  greenish-yellow  tint. 

Pathogenesis. — Glanders  occurs  principally  among  horses  and 
asses,  but  may  be  contracted  by  man  from  contact  with  infected 
animals  ;  it  has  also  been  communicated,  in  one  instance  with  a  fatal 
result,  by  subcutaneous  inoculation,  resulting  accidentally  from  the 
use  of  an  imperfectly  sterilized  hypodermic  syringe  which  had  pre- 
viously been  used  for  injecting  cultures  of  the  bacillus  into  guinea- 
pigs.  The  field  mouse  and  the  guinea-pig  are  especially  susceptible 
to  infection  by  experimental  inoculations  ;  the  cat  and  the  goat  may 
be  infected  in  the  same  way.  Lions  and  tigers  in  menageries  are 
said  to  have  contracted  glanders  from  being  fed  upon  the  flesh  of  in- 
fected animals  (Baumgarten).  Rabbits  have  but  slight  susceptibility, 
and  the  same  is  true  of  sheep  and  dogs ;  swine,  cattle,  white  mice, 
and  common  house  mice  are  immune. 

The  etiological  relation  of  the  bacillus  is  fully  established  by  the 
experiments  of  Loffler  and  Schiitz,  confirmed  by  other  bacteriologists, 
which  show  that  pure  cultures  injected  into  horses,  asses,  and  other 
susceptible  animals,  produce  genuine  glanders.  The  disease  is  char- 
acterized in  the  equine  genus  by  the  formation  of  ulcers  upon  the 
nasal  mucous  membrane,  which  have  irregular,  thickened  margins 
and  secrete  a  thin,  virulent  mucus ;  the  submaxillary  lymphatic 
glands  become  enlarged  and  form  a  tumor  which  is  often  lobulated  ; 
other  lymphatic  glands  become  inflamed,  and  some  of  them  suppurate 
and  open  externally,  leaving  deep,  open  ulcers ;  the  lungs  are  also 
involved  and  the  breathing  becomes  hurried  and  irregular.  In  farcy, 
which  is  a  more  chronic  form  of  the  same  disease,  circumscribed 
swellings,  varying  in  size  from  a  pea  to  a  hazelnut,  appear  on  differ- 
ent parts  of  the  body,  especially  where  the  skin  is  thinnest  ;  these 
suppurate  and  leave  angry-looking  ulcers  with  ragged  edges,  from 
which  there  is  an  abundant  purulent  discharge.  The  specific  bacillus 


492 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 


can  easily  be  obtained  in  pure  cultures  from  the  interior  of  suppurat- 
ing nodules  and  glands  which  have  not  yet  opened  to  the  surface, 
and  the  same  material  will  give  successful  results  when  inoculated 
into  susceptible  animals.  But  the  discharge  from  the  nostrils  or  from 
an  open  ulcer  contains  comparatively  few  bacilli ;  and  as  these  are 
associated  with  various  other  bacteria  which  grow  more  readily  in 
oar  culture  media,  it  is  not  easy  to  obtain  pure  cultures,  by  the  plate 
method,  from  such  material. 

In  the  guinea-pig  subcutaneous  inoculation  is  followed  in  four  or 
five  days  by  tumefaction  at  the  point  of  inoculation,  and  after  a  time 
a  prominent  tumor  with  caseous  contents  is  developed  ;  ulceratioii  of 
the  skin  follows,  and  a  chronic,  purulent  ulcer  with  irregular,  indu- 
rated margins  results ;  after  a  time  this  may  cicatrize.  Meanwhile 
the  lymphatic  glands  become  involved,  and  the  symptoms  of  general 


FIG.  123.— Section  through  a  glanders  nodule  in  liver  of  field  mouse.    Tissue  X  250.    Bacilli 
X  500.    (Baumgarten.) 

infection  are  developed  at  the  end  of  four  or  five  weeks  ;  the  glands 
suppurate,  and  in  males  the  testicles  are  also  involved  ;  finally  a  dif- 
fuse inflammation  of  the  joints  occurs,  and  death  results  from  ex- 
haustion. In  the  guinea-pig  the  specific  ulcers  upon  the  nasal  mu- 


BACILLI   IX   CHRONIC    INFECTIOUS   DISEASES.  493 

cous  membrane,  which  characterize  the  disease  in  the  horse,  are  rarely 
developed  to  any  great  extent. 

In  field  mice  general  infection  occurs  at  once  as  a  result  of  the 
subcutaneous  injection  of  a  small  quantity  of  a  pure  culture,  and  the 
animal  dies  at  the  end  of  three  or  four  days.  Upon  post-mortem 
examination  the  principal  changes  are  found  in  the  liver  and  in  the 
greatly  enlarged  spleen.  Scattered  through  these  organs  are  minute 
gray  points  which  are  scarcely  visible  to  the  naked  eye.  In  the 
guinea-pig,  which  succumbs  at  a  later  date,  these  nodules  are  larger 
and*  closely  resemble  miliary  tubercles,  both  macroscopically  and 
under  the  microscope,  in  stained  sections  of  the  tissues.  Similar 
nodules  are  also  found  in  the  kidneys  and  in  the  lungs  ;  they  have  a 
decided  tendency  to  undergo  purulent  degeneration.  The  bacilli  are 
found  principally  in  these  nodules,  of  recent  formation,  and  are  com- 
monly associated  in  groups,  as  if  they  had  been  enclosed  in  the  inte- 
rior of  a  cell  the  membranous  envelope  of  which  had  undergone 
degeneration  and  disappeared. 

As  before  remarked,  it  is  not  an  easy  matter  to  demonstrate  the 
bacillus  in  sections  of  the  tissues  containing  these  nodules,  owing  to 
the  facility  with  which  they  lose  their  color  in  alcohol  and  other  de- 
colorizing agents.  For  this  reason  it  will  be  best  to  dehydrate  sec- 
tions by  the  use  of  aniline  oil  (Weigert's  method)  or  to  resort  to 
Kuhne's  method  of  staining. 

It  is  also  difficult  to  demonstrate  the  presence  of  the  bacillus  in 
nodules  which  have  undergone  purulent  degeneration,  in  the  secre- 
tions from  the  nostrils  of  horses  suffering  from  glanders,  or  in  the 
pus  from  the  specific  ulcers  and  suppurating  glands  ;  for  they  are 
present  in  comparatively  small  numbers.  But  the  virulent  nature  of 
these  discharges  is  shown  by  inoculations  into  guinea-pigs  or  mice, 
and  it  is  easier  to  obtain  a  pure  culture  from  such  virulent  material 
by  first  inoculating  a  susceptible  animal  than  directly  by  the  plate 
method;  for  the  small  number  of  bacilli  present,  and  their  associa- 
tion with  other  bacteria  which  develop  more  rapidly  in  our  culture 
media,  make  this  a  very  uncertain  procedure.  For  establishing  the 
diagnosis  of  glanders,  therefore,  Loffler  recommends  the  inoculation 
of  guinea-pigs  with  pus  from  a  suppurating  gland  or  ulcer,  or  the 
nasal  discharge  from  a  suspected  animal,  rather  than  a  direct  attempt 
to  demonstrate  the  presence  of  the  bacillus  by  staining  and  culture 
methods. 

The  method  proposed  by  Strauss  gives  more  prompt  results. 
This  consists  in  the  intraperitoneal.  injection  of  cultures  or  of  the 
suspected  products  into  the  cavity  of  the  abdomen  of  male  guinea- 
pigs.  If  the  glanders  bacillus  is  present  tho  diagnosis  may  be  made 
within  three  or  four  days  from,  the  infectious  process  established  in 


494  BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES. 

the  testicles.  At  the  end  of  this  time  the  scrotum  is  red  and  shining, 
the  epidermis  desquamates,  and  suppuration  occurs,  the  pus  some- 
times perforating  the  integument.  This  pus  is  found  to  contain  the 
glanders  bacillus.  The  animal  usually  dies  in  the  course  of  twelve 
to  fifteen  days.  When  the  animals  are  killed  three  or  four  days 
after  the  inoculation,  the  two  layers  of  the  tunica  vaginalis  testis 
are  found  to  be  covered  with  a  purulent  exudate  containing  the 
glanders  bacillus  and  to  be  more  or  less  adherent.  Even  as  early 
as  the  second  day  the  tunica  vaginalis  is  seen  to  be  covered  with 
granulations. 

An  attenuation  of  virulence  occurs  in  cultures  whicn  have  been 
kept  for  some  time,  and  inoculations  with  such  cultures  may  give  a 
negative  result ;  or,  when  considerable  quantities  are  injected,  may 
produce  a  fatal  result  at  a  later  date  than  is  usual  when  small 
amounts  of  a  recent  culture  are  injected  into  susceptible  animals. 

Kalning,  Preusse,  and  Pearson  have  obtained  from  cultures  of 
the  glanders  bacillus  a  glycarin  extract  similar  to  the  crude  tubercu- 
lin of  Koch — mallein.  This,  when  injected  into  animals  suffering 
from  glanders,  gives  rise  to  a  considerable  elevation  of  temperature, 
and  it  is  used  as  a  means  of  diagnosis  in  cases  of  suspected  infection  in 
animals  in  which  the  usual  symptoms  have  not  yet  manifested  them- 
selves. The  value  of  the  test  has  been  demonstrated  by  numerous 
experiments. 

Bonome  (1894),  as  a  result  of  extended  researches,  arrives  at  the 
following  conclusions : 

"  1.  The  bacillus  is  found  not  only  in  the  diseased  tissues  and 
purulent  discharges,  but  also  in  the  urine  and  milk  of  infected  ani- 
mals. 

"  2.  The  bacillus  is  found  in  the  foetus  of  infected  animals  even 
when  the  placenta  is  free  from  any  pathological  change. 

"  3.  The  glanders  bacillus  is  very  sensitive  to  desiccation  and  will 
not  grow  after  being  preserved  for  ten  days  at  25°  C. 

"  4.  In  distilled  water  the  bacillus  dies  out  in  six  days. 

"  5.  On  the  contrary,  when  protected  from  desiccation  it  resists 
a  comparatively  high  temperature — 70°  C.  for  six  hours;  a  temper- 
ature of  90°  to  100°  C.  destroys  it  in  three  minutes." 

BACILLUS   OF   LUSTGARTEN. 

Synonym. — Syphilis  bacillus. 

Found  by  Lustgarten  (1884)  in  syphilitic  lesions  and  secretions  of  syphi- 
litic ulcers,  and  believed  by  him  to  be  the  specific  infectious  agent  in  this 
disease.  No  satisfactory  experimental  evidence  that  this  is  the  case  has  yet 
been  obtained. 

Morphology.— Straight  or  curved  bacilli,  which  bear  considerable  resem- 
blance to  tubercle  bacilli,  but  differ  from  them  in  the  staining  reactions. 
They  are  usually  more  or  less  curved,  or  bent  at  a  sharp  angle,  or  S-shaped ; 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  495 

the  ends  often  present  slight  knob -like  swellings  ;  the  length  is  from  three 
and  one-half  /"  to  four  and  one-half  /*,  and  the  diameter  is  from  0.25  to  0.3  /*. 
With  a  high  power  the  contour  is  seen  to  be  not  quite  regular,  but  wavy  in 
outline,  and  bright  shining  spaces  in  the  deeply  stained  rods  may  be  ob- 
served ;  these,  from  two  to  four  in  a  single  rod,  are  believed  by  Lustgarten 
to  be  spores.  The  bacilli  are  not  found  free  in  the  tissues,  but  are  enclosed 
in  cells  of  a  round-oval  or  polygonal  form,  which  are  said  to  be  about  double 
the  size  of  a  white  blood  corpuscle.  The  bacilli  are  riot  numerous,  and  very 
commonly  only  one  or  two  are  found  in  a  single  cell,  but  groups  of  six  or 
eight  may  sometimes  be  seen,  especially  upon  the  margins  of  a  s}rphilitic 
lesion,  and  in  the  tissues  in  the  immediate  vicinity  of  the  infiltration,  which 
show  out  little  change  or  are  apparently  healthy  (Lustgarten). 

The  presence  of  these  bacilli  in  syphilitic  lesions  was  demonstrated  by 
Lustgarten  by  the  following  staining  method :  The  thin  sections  are  placed 
in  the  Ehrlich-Weigert  gentian- violet  solution  (one  hundred  parts  aniline 
water,  eleven  parts  saturated  alcoholic  solution  of  gentian  violet)  for  from 
twelve  to  twenty-four  hours  at  the  room  temperature,  and  two  hours  in  the 
incubating  oven  at  40°  C.  The  sections  are  then  thoroughly  washed  in  alco- 
hol and  placed  for  ten  seconds  in  a  1.5-per-cent  solution  of  potassium  per- 
manganate; in  this  solution  a  precipitate  of  peroxide  of  manganese  is 


FIG.  134.  FIG.  125. 

FIG.  124.— Migrating  cell  containing  syphilis  bacilli.    (Lustgarten. ) 

Fia.  125.— Pus  from  hard  chancre  containing  syphilis  bacilli     (Lustgarten.') 

formed,  which  adheres  to  the  section  ;  this  is  dissolved  and  washed  off  in  a 
dilute  aqueous  solution  of  pure  sulphuric  acid;  the  sections  are  then  washed 
in  water,  and,  if  not  completely  decolorized,  are  returned  for  a  few  seconds  to 
the  permanganate  solution  and  again  washed  off  in  the  acid;  it  may  be 
necessary  to  repeat  this  operation  three  or  four  times.  Finally  the  sections 
are  dehydrated  and  mounted  in  balsam  in  the  usual  manner.  Cover-glass 
preparations  are  made  in  the  same  way,  except  that,  after  being  taken  from 
the  staining  solution,  they  are  washed  off  in  water  instead  of  in  alcohol. 

Another  method  of  staining,  recommended  by  De  Giacoma,  consists  in 
placing  the  sections  for  twenty -four  hours  in  aniline-water-fuchsin  solution 
(cover-glass  preparations  may  be  stained  in  the  same  solution,  hot,  in  a  few 
minutes),  then  washing  them  in  water,  and  decolorizing  in  a  solution  of  per- 
chloride  of  iron — first  in  a  dilute  and  then  in  a  saturated  solution. 

The  method  of  staining  employed  by  Lustgarten  serves  to  differentiate 
his  bacillus  from  many  other  microorganisms,  but  not  from,  the  tubercle  ba- 
cillus and  the  bacillus  of  leprosy,  which,  as  he  pointed  out,  may  be  stained 
in  the  same  way.  And  it  has  since  been  shown  by  Alvarez  and  Tavel,  and 
by  Matterstock,  that  in  smegma  from  the  prepuce  or  the  vulva,  bacilli  are 
found  which  have  the  same  staining  reaction  and  are  similar  in  their  mor- 
phology to  the  bacillus  of  Lustgarten.  This  by  no  means  proves  that  the 


496 


BACILLI   IN   CHRONIC    INFECTIOUS    DISEASES. 


smegma  bacilli  found  under  the  prepuce  of  healthy  persons  are  identical 
•with  the  bacilli  found  by  Lustgarten  and  others  in  sections  of  tissues  involved 
'in  syphilomata.  In  the  absence  of  pure  cultures  and  inoculation  experiments 
it  is  impossible  to  establish  identity,  however  similar  may  be  the  characters 
referred  to.  Several  well-known  pathogenic  bacilli  resemble  quite  as  closely 
in  these  particulars  other  bacilli  which  have,  nevertheless,  been  differentiated 
from  them  by  culture  and  inoculation  experiments.  We  may  mention 
especially  in  this  connection  the  bacillus  of  diphtheria,  as  obtained  from  the 
pseudo-membranous  exudation  in  a  genuine  case  of  this  disease,  and  the 
pseudo  diphtheria  bacilli  found  by  Roux  and  Yersinin  the  fauces  of  healthy 
children.  On  the  other  hand,  since  it  has  been  shown  that  similar  bacilli 
are  common  in  preputial  srnegma,  we  cannot  attach  great  importance  to  the 
finding  of  Lustgarten's  bacillus  in  primary  syphilitic  sores ;  and  it  has  not 
been  found  in  sufficient  numbers,  or  with  sufficient  constancy,  by  those  who 
have  searched  for  it  subsequently  to  the  publication  of  Lustgarten's  inves- 
tigations, to  give  strong  support  to  the  view  that  it  is  the  specific  infectious 
agent  in  syphilis.  Baumgarten,  who  has  searched  in  vain  for  Lustgarten's 
bacillus  in  uncomplicated  visceral  syphilomata,  suggests  that  the  bacilli 
found  occasionally  in  such  lesions  were  perhaps  tubercle  bacilli  and  repre- 
sented a  mixed  infection.  As  the  bacillus  under  consideration  has  not  been 
obtained  in  cultures,  we  have  no  information  as  to  its  biological  characters 
and  pathogenesis. 


BACILLUS    OF   RHINOSCLEROMA. 

First  observed  by  Von  Frisch  (1882)  in  the  newly  formed  tubercles  of 
rhinoscleroma.  Cultivated  by  Paltauf  a'nd  Von  Eiselberg  (1880). 

Rhinoscleroma  is  a  chronic  affection  of  the  skin,  and  especially  of  the 
mucous  membrane  of  the  nares,  which  is  characterized  by  the  formation  of 
tubercular  thickenings  of  the  skin  and  tumefaction  of  the  nasal  mucous 
membrane,  followed  sometimes  by  ulceratiori  It  prevails  in  Italy,  Austria, 
and  to  a  slight  extent  in  some  parts  of  Germany.  Pathologists  generally 
regard  it  as  an  infectious  process,  although  this  has  not  been  proved. 

The  bacilli,  first  described  by  Von  Frisch,  appear  to  be  constantly  present 
in  the  newly  formed  tubercles.  They  are  commonly  found  in  certain  large 


FIG.  126.— Bacillus  of  rhinoscleroma  iii  lymphatic  vessels  of  the  superficial  part   of  tumor. 
x  1,200.    (Cornil  and  Babes  ) 

hyaline  cells  peculiar  to  the  disease,  and  may  also  be  observed  in  the  lym- 
phatic vessels  or  scattered  about  in  the  involved  tissues. 

Morphology. — Short  bacilli  with  rounded  ends,  usually  united  in  pairs, 
and  surrounded  by  a  gelatinous  capsule  resembling  that  of  Friedlander's 


BACILLI   IN   CHRONIC   INFECTIOUS   DISEASES.  497 

bacillus.     According  to  Eiseiiberg,  the  bacilli  are  two  to  three  times  as  long 
as  broad,  and  may  grow  out  into  filaments. 

These  bacilli  stain  readily  with  the  aniline  colors  and  by  Gram's  method. 
The  capsule  may  be  demonstrated  by  the  methods  usually  employed  in  stain- 
ing Friedlander's  bacillus,  or  by  the  following  method  which  is  especially 
recommended  by  Alvarez :  The  excised  portions  of  tissue  involved  in  the  dis- 
ease are  placed  for  twenty-four  hours  in  a  one-per-cent  solution  of  osmic 
acid  and  then  in  absolute  alcohol.  When  properly  hardened  thin  sections 
are  made;  these  are  stained  in  a  hot  solution  of  aniline-water-methyl-violet 
for  a  few  minutes,  and  then  decolorized,  by  Gram's  method,  in  iodine  so- 
lution. 

Biological  Characters. — An  aerobic,  non-motile,  non-liquefying  bacillus 
(facultative  anaerobic  ?). 

In  gelatin  stab  cultures  the  growth  resembles  that  of  Friedlander's  ba- 
cillus— i.e.,  a  nail-like  growth,  consisting  of  densely  crowded,  opaque  colonies 
along  the  line  of  puncture,  and  a  heaped-up,  white,  glistening  mass  upon  the 
surface,  hemispherical  in  form  and  viscous  in  consistence.  Upon  gelatin 
plates  yellowish-white,  spherical  colonies  are  developed  within  two  or  three 
days,  which  under  the  microscope  are  seen  to  be  granular.  Upon  potato  a 
cream-like  growth  occurs  along  the  line  of  inoculation,  which  is  white  or 
yellowish-white  in  color,  and  in  which  gas  bubbles  may  be  developed.  De- 
velopment is  most  rapid  at  a  temperature  of  35°  to  38°,  but  also  occurs  at  the 
room  temperature.  • 

Pathogenesis. — The  etiological  relation  of  this  bacillus  to  the  disease  with 
which  it  is  associated  has  not  been  established.  It  is  pathogenic  for  mice 
and  for  guinea-pigs,  less  so  for  rabbits;  in  this  regard,  as  in  its  morphology 
and  growth  in  various  culture  media,  it  bears  a  close  resemblance  to  Fried- 
lander's  bacillus,  which  is  also  found  not  infrequently  in  the  nasal  secretions 
of  healthy  persons  and  in  those  suffering  from  chronic  nasal  catarrh  or  ozaena. 

The  principal  points  of  difference,  as  pointed  out  by  Baumgarten,  are  as 
follows :  The  bacillus  of  rhinoscleroma  is  usually  more  decidedly  rod-shaped 
than  Friedlander's  bacillus,  although  both  may  be  of  so  short  an  oval  as  to 
resemble  micrococci.  The  first-mentioned  bacillus  constantly  presents  the 
appearance  of  being  surrounded  by  a  transparent  capsule,  even  in  the  cul- 
tures in  artificial  media, .while  Friedlander's  bacillus  in  such  media  does  not 
usually  present  this  appearance,  unless  as  a  result  of  special  treatment. 
Finally,  the  bacillus  of  rhinoscleroma  may  retain  its  color,  in  part  at  least, 
when  treated  by  Gram's  method,  while  Friedlander's  bacillus  is  completely 
decolorized  when  placed  in  the  iodine  solution  employed  in  this  method. 

Notwithstanding  these  points  of  difference,  Baumgarten  is  not  entirely 
satisfied  that  this  bacillus  is  a  distinct  species,  and  several  bacteriologists 
have  maintained  that  it  is  identical  with  the  bacillus  of  Friedlander. 

32 


XIII. 

BACILLI  WHICH  PRODUCE  SEPTIC^MIA 
SUSCEPTIBLE  ANIMALS. 

WHEN,  as  a  result  of  accidental  (natural)  or  experimental  inocula- 
tion, a  microorganism  is  introduced  into  the  body  of  a  susceptible 
animal  which  is  able  to  multiply  in  its  blood,  producing  a  general  in- 
fection, we  speak  of  this  general  blood  infection  as  a  septicaemia. 
When  pathogenic  microorganisms  which  are  unable  to  multiply  in 
the  blood  establish  themselves  in  some  particular  locality  in  the  ani- 
mal body  which  is  favorable  for  their  growth,  and  by  the  formation 
of  toxic  products,  which  are  absorbed,  give  rise  to  general  symptoms 
of  poisoning,  we  designate  the  affection  toxcemia.  As  examples  of 
this  mode  of  pathogenic  action  we  may  mention  diphtheria  and 
tetanus.  As  a  rule,  the  various  forms  of  septicaemia  are  quickly 
fatal,  and,  as  the  microorganisms  to  which  they  are  due  multiply  in 
the  blood  of  the  infected  animal,  this  fluid  possesses  infectious  pro- 
perties, and,  when  inoculated  in  the  smallest  quantity  into  another 
susceptible  animal,  reproduces  the  same  morbid  phenomena.  A  typi- 
cal example  of  this  class  of  diseases  is  found  in  anthrax,  to  which 
disease  a  special  section  has  already  been  devoted  (VIII.).  But  in 
this  and  other  forms  of  septicaemia  subcutaneous  inoculations  do  not, 
as  a  rule,  result  in  the  immediate  invasion  of  the  blood  by  the  para- 
sitic microorganism.  Often  a  local  inflammatory  process  of  consider- 
able extent  is  first  induced  ;  and  in  some  cases  general  infection  only 
occurs  a  short  time  before  the  death  of  the  animal,  depending,  per- 
haps, upon  a  previous  toxaemia  from  the  absorption  of  toxic  products 
developed  at  the  seat  of  local  infection.  The  pathogenic  action,  then, 
in  acute  forms  of  septicaemia  appears  to  result,  not  alone  from  the 
presence  and  multiplication  of  the  pathogenic  microorganism  in  the 
blood,  but  also  from  the  toxic  action  of  products  evolved  during  its 
growth. 

Some  of  the  pathogenic  bacilli  of  this  class  now  known  to  bac- 
teriologists have  been  discovered  by  studying  the  infectious  diseases 
induced  by  them  in  lower  animals  among  which  these  diseases  pre- 
vail naturally — i.e.,  independently  of  human  interference.  Many 


BACILLI    WHICH    PRODUCE   SEPTICAEMIA.  4(J'J 

more  are  known  to  us  from  experiments  made  in  pathological  labora- 
tories, in  testing  by  inoculations  into  animals  bacteria  obtained  from 
various  sources,  with  reference  to  their  pathogenic  power.  We  in- 
clude in  this  group  only  those  bacilli  which  induce  fatal  septicaemia 
in  susceptible  animals  when  injected  into  the  circulation  or  sub- 
cutaneously  in  a  comparatively  small  quantity — e.g.,  less  than  half 
a  cubic  centimetre  of  a  bouillon  culture. 

BACILLUS   SEPTICAEMIA   H^EMORRHAGIC^E. 

Synonyms. — Bacillus  of  fowl  cholera ;  Microbe  du  cholera  des 
poules  (Pasteur) ;  Bacillus  choleras  gallinarum  (Fliigge) ;  Bacillus  der 
Hiihnercholera ;  Bacillus  of  rabbit  septicaemia  ;  Bacillus  cuniculi- 
cida  (Fliigge)  ;  Bacillus  der  Kaninchenseptikamie  (Koch)  ;  Bacillus 
der  Rinderseuche  (Kitt)  ;  Bacillus  der  Schweineseuche  (Loffler  and 
Schiitz)  ;  Bacillus  der  Wildseuche  (Hueppe)  ;  Bacillus  der  Biiffel- 
seuche  (Oreste-Armanni)  ;  (Bacterium  of  Davaine's  septicaemia  ?) 

It  is  now  generally  admitted  by  bacteriologists  that  Koch's  ba- 
cillus of  rabbit  septicaemia  (1881)  is  identical  with  the  bacillus 
("micrococcus")  of  fowl  cholera  previously  described  by  Pasteur 
(1880).  The  similar  bacilli  found  in  the  blood  of  animals  dead  from 
the  infectious  diseases  known  in  Germany  as  Wildseuche  (Hueppe), 
Rinderseuche  (Kitt),  Schweineseuche  (Schiitz),  and  Biiffelseuche 
(Oreste-Armanni)  appear  also  to  be  identical  with  the  bacillus  of 
rabbit  septicaemia  and  fowl  cholera.  This  view  is  sustained  by 
Hueppe  and  by  Baumgarten,  and  by  the  comparative  researches  of 
Caneva  (1891)  and  of  Bunzl-Federn  (1891). 

This  is  evidently  a  widely  distributed  pathogenic  bacillus  ;  it  was 
obtained  by  Koch  from  rabbits  inoculated  with  pu- 
trefying flesh  infusion,  by  Gaffky  from  impure  river 

water,  andbv  Pasteur  from  the  blood  of  fowls  suffer- 

.          .  . 

ing  from  the  infectious  disease  known  in  France  as    ~y~\ 


cholera  des  poules.     It  is  not  infrequently  found  in    „  ^Q  V*O  ' 
putrefying  blood,   and  its  presence  in  the  salivary   '{j  *'% 
secretions  of  man  has  occasionally  been  demonstrated     F     197  _Bacillua 

(Baumgarten).  septicaemia)   hsemor- 

With   reference  to  the  American   swine   plague  rhagica?  mthet 

*      &         of  a   rabbit,    x  950. 

described  by  Salmon  and  Smith,  we  are  informed  by  (Baumgarten.) 
Smith,  in  his  most  recent  publication  upon  the  subject 
(Zeitschrift  fur  Hygiene,  Band  x.,  page  493),  that  cultures  of  the 
German  Schweineseuche  bacillus,  received  from  the  Berlin  Hygienic 
Institute,  compared  with  his  cultures  from  infected  swine  in  this 
country,  agreed  in  all  particulars,  except  that  the  former  were  de- 
cidedly more  pathogenic  for  swine  and  for  rabbits. 

It  appears  extremely  probable  that  the  form  of  septicaemia  studied 


500  BACILLI    WHICH    PRODUCE   SEPTIC^MIA 

by  Davaine  (1872),  which  he  induced  in  the  first  instance  by  inject- 
ing putrid  ox  blood  into  rabbits,  was  due  to  the  same  pathogenic  ba- 
cillus. The  writer  obtained  this  bacillus  (1887)  in  Cuba  from  the 
blood  of  rabbits  inoculated  with  liver  tissue  taken  from  a  yellow- 
fever  cadaver  and  kept  for  forty-eight  hours  in  an  antiseptic  wrap- 
ping. The  name  which  we  have  adopted  is  that  proposed  by  Hueppe 
for  the  form  of  septicaemia  to  which  it  gives  rise — "Septikamia 
hamorrhagica. " 

Morphology. — Short  bacilli  with  rounded  ends,  from  0.6  to  0.7 
/*  in  diameter  and  about  1.4  ^  long;  sometimes  united  in  pairs,  or 
in  chains  of  three  or  four  elements.  In  stained  preparations  the  ex- 
tremities are  usually  stained,  while  the  central  portion  of  the  rod 
remains  unstained.  This  "  end  staining"  causes  the  rods  to  present 
the  appearance  of  diplococci  when  examined  with  a  comparatively 
low  power,  and  some  of  the  earlier  observers  described  the  microor- 
ganism under  consideration  as  a  micrococcus.  It  is  quickly  stained 
by  the  aniline  colors  usually  employed,  but  loses  its  color  when 
treated  by  Gram's  method. 

Biological  Characters. — A  non-motile,  aerobic,  non-liquefy- 
ing bacillus.  Does  not  form  spores.  Grows  in  various  culture  media 
at  the  room  temperature,  but  more  rapidly  at  35°  to  37°  C. — the 
lowest  temperature  at  which  development  occurs  is  about  13°  C. 
Although  this  is  an  aerobic  bacillus  and  a  certain  amount  of  oxygen 
is  necessary  for  its  development,  it  appears  to  grow  better  when  the 
amount  is  somewhat  restricted  than  it  does  on  the  surface  of  nutrient 
media. 

Upon  gelatin  plates,  at  the  end  of  two  or  three  days,  small, 
white  colonies  are  developed  upon  or  near  the  surface ;  these  are 
finely  granular  and  spherical,  with  a  more  or  less  irregular  outline, 
and  by  transmitted  light  have  a  yellowish  color  ;  later  the  central 
portion  of  the  colonies  is  of  a  yellowish-brown  color  and  is  sur- 
rounded by  a  transparent  peripheral  zone.  The  superficial  colonies 
are  commonly  smaller  than  those  which  develop  a  little  below  the 
surface  of  the  gelatin.  In  stab  cultures  in  nutrient  gelatin  the 
growth  upon  the  surface  consists  of  a  thin,  whitish  layer  in  the 
vicinity  of  the  point  of  puncture,  having  an  irregular,  jagged  out- 
line— sometimes  there  is  no  development  upon  the  surface ;  along 
the  line  of  puncture  the  growth  consists  of  rather  transparent,  dis- 
crete or  confluent  colonies.  In  streak  cultures  upon  nutrient  agar, 
or  gelatin,  or  blood  serum  the  growth  is  limited  to  the  immediate 
vicinity  of  the  line  of  inoculation,  and  consists  of  finely  granular, 
semi-transparent  colonies,  which  form  a  thin,  grayish-white  layer 
with  irregular,  somewhat  thickened  margins.  Upon  potato  no  de- 
velopment occurs,  as  a  rule,  at  the  room  temperature,  but  in  the  in- 


IN   SUSCEPTIBLE   ANIMALS. 


501 


>- 


cubating  oven  a  rather  thin,  transparent,  grayish-white  or  yellowish, 
waxy  layer  is  developed  in  the  course  of  a  few  days.  According  to 
Bunzl-Federn,  the  bacillus  of  fowl  cholera  and  that 
of  rabbit  septica3mia  grow  upon  potato,  while  the 
bacillus  of  Wildseuche,  Schweineseuche,  and  Biif- 
felseuche  do  not.  According  to 
Caneva,  none  of  the  bacilli  of  this 
group  grow  upon  potato.  The 
same  author  states  that  the  growth 
in  milk  is  scanty  and  does  not 
produce  coagulation,  while  Bunzl- 
Federn  finds  that  the  bacillus  of 
fowl  cholera  and  of  rabbit  septi- 
caemia produce  coagulation  and 
the  others  do  not.  These  differ- 
ences are  not,  however,  consid- 
ered by  the  author  last  named  as 
sufficient  to  establish  the  specific 
difference  of  the  bacilli  from  these 
different  sources.  He  looks  upon 
them  rather  as  varieties  of  the 
same  species.  Bunzl-Federn  has 
also  ascertained  that  when  cul- 
tivated in  a  peptone  solution  all 
of  the  bacilli  of  this  group,  with 
the  exception  of  that  obtained 
from  the  so-called  Buffelseuche, 
give  the  reaction  for  phenol  and 
for  indol — the  bacillus  of  Buffel- 
seuche gives  the  indol  reaction  only.  Development  in  bouillon  is  rapid 
and  causes  a  uniform  turbidity  of  the  fluid.  Cultures  of  this  bacillus 
may  retain  their  vitality  for  three  months  or 
more  when  kept  in  a  moist  condition ;  but 
the  bacillus  usually  fails  to  grow  after  having 
been  kept  for  a  few  days  in  a  desiccated  con- 
dition ;  according  to  Hueppe,  it  may  resist 
desiccation  for  fourteen  days.  The  thermal 
death-point,  as  determined  by  Salmon  for 
the  bacillus  of  fowl  cholera,  is  56°  C. ,  the  time 
of  exposure  being  ten  minutes  (55°  O.  with 
fifteen  minutes'  exposure — Baumgarten).  It 
is  not  readily  destroyed  by  putrefaction  (Kitt) . 
A  solution  of  mercuric  chloride  of  1  : 5,000 
destroys  it  in  one  minute,  and  a  three-per-cent  solution  of  -carbolic 


FIG.  128.  —  Bacillus 
septicaemias  haemor- 
rhagicae;  stick  culture 
in  nutrient  gelatin, 
end  of  four  days  at  16°- 
18°  C.  (Baumgarten  ) 


FIG.  129.— Bacillus 
of  Schweineseuche  ; 
old  stick  culture 
in  nutrient  gela- 
tin. (Schutz.) 


FIG.  130.  —  Bacillus  of  swine 
plague;  colonies  on  gelatin 
plate,  fciid  of  sov^a  days. 
X  60.  (Smith.) 


502  BACILLI   WHICH    PRODUCE    SEPTICAEMIA 

acid  in  six  hours  (Hueppe).  Pasteur  (1880)  has  shown  that  when 
cultures  of  this  bacillus  (microbe  of  fowl  cholera)  in  bouillon  are 
kept  for  some  time  they  gradually  lose  their  pathogenic  virulence, 
and  he  has  ascribed  this  "attenuation  of  virulence"  to  the  action  of 
atmospheric  oxygen.  He  also  ascertained  that  the  particular  degree 
of  virulence  manifested  by  the  mother  culture  after  a  certain  interval 
could  be  maintained  in  successive  cultures  made  at  short  intervals. 
He  was  thus  able  to  cultivate  different  pathogenic  varieties,  and  to 
use  these  in  making  protective  inoculations,  by  which  susceptible  ani- 


^ 


f:         ^y.>         -.      '.;: 

V  •  •    ""  •'*.    i  "  •  -•-     *  »«     ,   ""X. 

^v...  ..       •        „*.  ..-.    ».,..-..'  .••       *  .  rt         j, 

«*>'  **.'•'.,-•,    "\  ••  •  '»  .  *»«  .-         *-*       ':"\ 

*^  **    ^         :  s  .  -•'  ,  .  «•„  ••         M 

^    /f" •  ;\  :  ** ••;-  "•;;-•,-'--'.-  -"••'v^::     f 

.    «*.,;'.  .,•'•  &  -—':'  h^ 

<          ->  *••••:_;.--•'• 

I 

FIG.  131.— Bacillus  of  Scliweineseuche,  in  blood  of  rabbit.    (Schiitz.) 

mals  were  preserved  from  the  effects  of  virulent  cultures  injected 
subsequently. 

Attenuated  cultures  recover  their  virulence  when  inoculated  into 
very  susceptible  animals.  Thus  a  culture  which  would  produce  a 
non-fatal  and  protective  attack  in  a  chicken  may,  according  to  Pas- 
teur, kill  a  small  bird,  like  a  sparrow;  and  by  successive  inoculations 
from  one  sparrow  to  another  the  original  degree  of  virulence  may  be 
restored,  so  that  a  minute  quantity  of  a  pure  culture  would  be  f  aid 
to  a  chicken. 

Pathogenesis. — Pathogenic  for  chickens,  pigeons,  pheasants, 
sparrows,  and  other  small  birds,  for  rabbits  and  mice,  also  for  swine 
(Sehweineseuche),  for  cattle  (Rinderseuche),  and  for  deer  (Will- 
seuche).  (See  supra,  pp.  285-287.) 

The  researches  of  Smith  and  of  Moore  show  that  "  an  attenuated 
variety  of  bacteria,  belonging  to  the  group  of  swine-plague  bacteria 
and  not  distinguishable  from  them,  inhabit  the  mouth  and  upper  air 


IN   SUSCEPTIBLE   ANIMALS.  503 

passages  of  such  domesticated  animals   as   cattle,  dogs,  and  cats" 
(Smith). 

BACILLUS   OF   CHOLERA   IN   DUCKS. 

Obtained  by  Cornil  and  Toupet  (1888)  from  the  blood  of  ducks,  in  the 
Jardin  d'Acclhnation  at  Paris,  which  had  died  of  an  epidemic  disease  charac- 
terized by  diarrhoaa,  feebleness,  and  muscular  tremors,  and  which  resulted 
fatally  in  two  or  three  days. 

Morphology. — Does  not  differ  in  its  morphology  from  the  bacillus  of 
fowl  cholera  (Bacillus  septicaemias  hsemorrhagicae) ;  short  rods  with  rounded 
ends,  from  1  to  1.5  11  in  length  and  0.5  ju  broad. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method ;  the  ends 
stain  more  deeply  than  the  central  portion. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motilebacillus. 
Does  not  form  spores.  Grows  in  the  usual  culture  media  at  the  room  tem- 
perature. In  its  growth  in  various  media,  as  well  as  in  its  morphology,  Cornil 
and  Toupet  found  this  bacillus  to  correspond  with  the  bacillus  of  fowl 
cholera.  In  gelatin  stab  cultures  the  growth  upon  the  surface  consists  of  a 
thin,  grayish  layer,  and  along  the  line  of  puncture  as  small,  semi  transpa- 
rent, slightly  yellowish,  spherical  colonies.  Upon  agar,  in  the  incubating 
oven,  at  the  end  of  twelve  hours  small,  lentil  shaped,  waxy  colonies  are 
formed,  which  later  may  have  a  diameter  of  three  to  four  millimetres. 
Upon  potato  circular,  yellowish  colonies  are  formed,  which  become  con- 
fluent and  form  a  somewhat  depressed,  pale-yellow  layer. 

Pathogenesis. — According  to  Cornil  and  Toupet,  this  bacillus  is  patho- 
genic for  ducks,  but  not  for  chickens  or  pigeons,  and  only  kills  rabbits  when 
injected  in  considerable  quantity.  Ducks  die  in  from  one  to  three  days 
from  subcutaneous  injections,  or  by  the  ingestion  of  food  to  which  the  bacil- 
lus has  been  added. 

BACILLUS  OF  HOG  CHOLERA  (Salmon  and  Smith). 

Synonyms. — Bacillus  of  swine  plague  (Billings) ;  Bacillus  of  swine- 
pest  (Selaiider). 

According  to  Smith,  this  bacillus  was  first  described  by  Klein 
(1884) ;  it  was  first  obtained  in  pure  cultures  and  its  principal  char- 
acters determined  by  Salmon  and  Smith  (1885),  and  has  since  been 
studied  in  cultures  and  by  experimental  inoculations  by  Selander, 
Billings,  Frosch,  Welch,  Caneva,  Bunzl-Federii,  and  others. 

The  bacillus  is  found  in  the  blood  and  various  organs  of  hogs 
which  have  succumbed  to  the  infectious  disease  known  in  this  country 
as  hog  cholera  ;  and  also  in  the  contents  of  the  intestine,  from  which 
it  may  be  obtained  by  inoculations  into  rabbits,  but  is  not  easily  iso- 
lated by  the  plate  method  owing  to  the  large  number  of  other  bac- 
teria present  (Smith). 

Morphology. — Short  bacilli  with  rounded  ends,  1.2  to  1.5  /*  in 
length  and  0. 6  to  0. 7  /*  in  breadth  ;  usually  united  in  pairs. 

This  bacillus  is  easily  stained  by  the  aniline  colors  usually  em- 
ployed, but  does  not  retain  its  color  when  treated  by  Gram's  method. 
When  the  staining  agent  is  allowed  to  act  for  a  very  short  time  the 
ends  of  the  rods  may  be  stained  while  the  central  portion  remains 
unstained. 


504 


BACILLI   WHICH   PRODUCE   SEPTICAEMIA 


Biological  Characters. — An  aerobic  (facultative  anaerobic),  non- 
liquefying,  actively  motile  bacillus.  In  many  of  its  characters  this 
bacillus  closely  resembles  the  one  last  described  (Bacillus  septicaBmise 
hsemorrhagicse),  but  it  is  distinguished  from  it  by  its  active  move- 
ments, which,  according  to  Smith,  may  be  still  observed  in  cultures 
which  have  been  kept  for  weeks  or  months.  Does  not  form  spores. 
Grows  readily  in  various  culture  media  at  the  room  temperature — 
more  rapidly  in  the  incubating  oven.  Upon  gelatin  plates  colonies 
are  developed  in  from  twenty-four  to  forty-eight  hours.  The  deep  colo- 
nies are  spherical  and  homogeneous,  and  have  a  brownish  color  by 
transmitted  light;  they  seldom  exceed  one-half  millimetre  in  diameter. 


FIG.  132.— Bacillus  of  hog  cholera;  stained  by  Loffler's  method  to  show  flagella.    x  1,000.  From 
a  photomicrograph  made  at  the  Army  Medical  Museum.    (Gray.) 

The  superficial  colonies  may  attain  a  diameter  of  two  millimetres ; 
they  present  no  distinctive  characters.  Upon  agar  plates  the  colonies 
may  have  a  diameter  of  four  millimetres  ;  they  have  a  grayish,  trans- 
parent appearance  and  a  shining  surface.  In  gelatin  stab  cultures 
small,  yellowish-white  colonies  are  developed  along  the  line  of  in- 
oculation, which  may  become  confluent ;  upon  the  surface  a  thin, 
pearly  layer  is  developed  about  the  point  of  inoculation,  which  may 
have  a  diameter  of  six  millimetres  or  more.  Upon  potato  a  straw- 
yellow  layer  is  developed,  which  later  acquires  a  darker  color.  In 
slightly  alkaline  bouillon  a  slight  cloudiness  may  be  observed  at  the 
end  of  twenty-four  hours,  and  at  the  end  of  one  or  two  weeks,  if 
not  disturbed,  a  deposit  is  seen  at  the  bottom  of  the  tube  and  a  thin, 
broken  film  may  form  upon  the  surface.  The  development  of  this 
bacillus  in  milk  produces  a  direct  solution  of  the  casein  without  pre- 
vious coagulation  ;  when  a  solution  of  litmus  has  been  added  to  milk 


IN   SUSCEPTIBLE   ANIMALS.  505 

it  retains  its  blue  color  in  presence  of  this  bacillus,  while  the  bacillus 
previously  described  causes  it  to  change  to  red.  Neither  phenol 
nor  indol  is  produced  in  solutions  containing  peptone  (Bunzl-Federn) 
—another  distinguishing  character  from  the  Bacillus  septicsemise 
haemorrhagiccE.  This  bacillus  may  be  cultivated  in  slightly  acid 
media,  which  after  a  time  acquire  an  alkaline  reaction. 

In  Smith's  experiments  this  bacillus  was  found  to  resist  desicca- 
tion from  nine  days  to  several  months,  according  to  the  thickness  of 
the  layer  dried  upon  the  cover  glass  ;  bacilli  from  an  agar  culture  in 
some  experiments  failed  to  grow  after  seventeen  days,  and  in  others 
still  gave  cultures  after  four  months.  Bouillon  cultures  are  steril- 
ized in  four  minutes  by  a  temperature  of  70°  C.,  in  fifteen  minutes 
by  58°  C.,  and  in  one  hour  by  54°  C.  (Smith).  Novy  has  isolated 
from  cultures  of  the  hog-cholera  bacillus  a  toxic  basic  substance 
which  he  calls  susotoxin.  This  was  obtained  by  Brieger's  method  ; 
it  is  a  yellowish-brown,  syrup-like  liquid,  which,  when  injected  into 
rats  in  doses  of  0.125  to  0.25  cubic  centimetre,  causes  their  death  in 
less  than  thirty-six  hours.  He  also  obtained  by  precipitation  with 
absolute  alcohol,  from  cultures  concentrated  in  a  vacuum  at  36°  C., 
a  toxalbumin  which  when  dried  was  in  the  form  of  a  white  powder 
easily  soluble  in  water.  Rats  died  in  three  or  four  hours  after  re- 
ceiving subcutaneously  a  dose  of  0. 1  to  0. 5  gramme. 

Pathogenesis. — Pathogenic  for  swine,  rabbits,  guinea-pigs,  mice, 
and  pigeons. 

In  certain  parts  of  the  United  States  the  disease  known  as  "  hog 
cholera  "  frequently  prevails  among  swine  as  a  fatal  epidemic.  It 
may  occur  as  an  acute  and  quickly  fatal  septicaemia,  or  in  a  more 
chronic  form  lasting  from  two  to  four  weeks  or  even  longer.  In 
the  acute  form  death  may  occur  within  twenty-four  hours,  and  hsem- 
orrhagic  extravasations  are  found  upon  the  mucous  and  serous 
membranes  and  in  the  parenchyma  of  the  lungs,  kidneys,  and  lym- 
phatic glands.  The  spleen  is  greatly  enlarged,  soft,  and  dark  in 
color.  In  the  chronic  form  of  the  disease  the  most  notable  changes 
are  found  in  the  alimentary  canal.  These  are  most  constant  and 
characteristic  in  the  caecum  and  colon,  which  may  be  studded  with 
spherical,  hard,  necrotic  masses  or  extensive  diphtheritic  patches. 
According  to  Smith,  the  hsemorrhagic  and  necrotic  form  of  the  dis- 
ease may  exist  at  the  same  time  in  different  animals  of  the  same 
herd.  The  bacilli  are  found  in  all  of  the  organs,  and  especially  in 
the  spleen,  where  they  are  associated  in  irregular  colonies  similar 
to  those  of  the  typhoid  bacillus.  Smith  has  demonstrated  their  pre- 
sence in  urine  taken  from  the  bladder  immediately  after  the  death 
of  the  animal,  and  states  that  the  kidneys  are  almost  always  in- 


BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

volved,  as  shown  by  the  presence  of  albumin  and  tube  casts  in  the 
urine. 

An  extremely  minute  quantity  of  a  bouillon  culture  injected  be- 
neath the  skin  of  a  rabbit  causes  its  death  in  from  seven  to  twelve 
days  ;  a  larger  quantity  may  produce  a  fatal  result  in  five  days  ;  in- 
travenous injections  of  very  small  amounts  may  be  fatal  within 
forty-eight  hours.  After  a  subcutaneous  injection  the  animal  re- 
mains in  apparent  good  health  for  three  or  four  days,  after  which  it 
loses  its  appetite  and  is  indisposed  to  move  ;  several  days  before 
death  the  temperature  is  suddenly  elevated  from  2°  to  3°  C.,  and  it 
remains  high  until  the  fatal  termination.  At  the  autopsy  the  spleen 
is  found  to  be  enlarged  and  of  a  dark-red  color  ;  the  liver  is  studded 
with  small,  yellowish- white,  necrotic  foci;  the  kidneys  have  under- 
gone parenchymatous  changes  ;  the  heart  is  fatty  ;  and  the  intestinal 
mucous  membrane  is  more  or  less  marked  with  haemorrhagic  extra- 
vasations. The  bacilli  are  found  in  all  of  the  organs.  In  house 
mice  the  results  of  experimental  inoculations  are  similar  to  those  in 
rabbits.  Guinea-pigs  succumb  when  inoculated  subcutaneously  with 
one-tenth  cubic  centimetre ;  pigeons  require  a  still  larger  dose — 
about  three-quarters  of  a  cubic  centimetre.  Swine  are  killed  by  the 
intravenous  injection  of  one  to  two  cubic  centimetres  of  a  recent 
bouillon  culture,  but,  as  a  rule,  do  not  succumb  to  subcutaneous 
injections.  Cultures  recently  obtained  from  diseased  animals  are 
more  virulent  than  those  which  have  been  propagated  for  a  consider- 
able time  in  artificial  media. 

Smith  has  described  a  variety  of  the  hog-cholera  bacillus  obtained  during- 
an  epidemic  in  which  the  disease  was  of  longer  duration — about  four  weeks 
— than  is  usual,  and  in  which,  there  was  commonly  found  at  the  autopsy  a 
diphtheritic  inflammation  of  the  mucous  membrane  of  the  stomach.  This 
bacillus  differed  from  the  typical  form  by  being  somewhat  larger  and  in 
forming  considerably  larger  colonies  in  gelatin  plates — two  or  three  times 
as  large.  It  also  produced  a  greater  opacity  in  peptonized  bouillon,  and  in 
general  showed  a  more  vigorous  growth  in  various  nutrient  media.  It  dif- 
fered also  in  its  pathogenic  power,  as  tested  upon  rabbits,  causing  death  at  a 
later  date  or  not  at  all ;  and  in  fatal  cases  the  swelling  of  the  spleen  and 
necrotic  foci  in  the  liver,  produced  by  the  first-described  species,  were  absent. 

Bang  (1892)  has  obtained  a  bacillus  from  infected  swine  in  Denmark 
which  corresponds  with  the  American  hog-cholera  bacillus.  In  chronic 
forms  of  the  disease  pneumonia  and  an  extensive  diphtheritic  process  in  the 
intestines  occurred  as  a  complication.  This  was  found  to  be  due  to  another 
bacillus,  called  by  Bang  "vacuole-bacillus."  This  produced  a  fatal  pleuro- 
pneumonia  when  injected  into  the  lungs  in  pigs.  According  to  Bang,  his 
"vacuole-bacillus"  is  without  doubt  identical  with  the  swine-plague  bacillus 
of  Salmon  and  Smith,  and  the  disease  of  swine  studied  by  him  was  a  mixed 
infection.  The  necrotic  changes  in  the  intestine,  found  in  cases  running  a 
chronic  course,  are  believed  by  Bang  to  be  due  to  still  another  bacillus — his 
"  necrosis-bacillus."  Affanassieff  (1892)  confirms  the  results  previously  ob- 
tained by  several  independent  observers  as  to  the  identity  of  the  swine-plague 
bacillus  of  Salmon  and  Smith  with  the  LofBer-Schiitz  bacillus.  The  only 
difference  observed  was  a  difference  in  pathogenic  virulence — the  bacillus 


IN   SUSCEPTIBLE   ANIMALS.  507 

from  America  corresponding-  with  a  somewhat  attenuated  variety  of  that 
from  Germany. 

Welch  (1894),  as  a  result  of  his  extended  researches,  arrives  at  the  follow- 
ing1 conclusion : 

"Our  own  conclusion  as  to  the  bacteria  of  Schweineseuche  and  of  swine 
plague  is  that  no  difference  exists  between  them  as  regards  morphology, 
culture  behavior,  and  pathogenic  effects  on  rabbits,  mice,  and  other  labora- 
tory animals.  Cultures  of  each  occur  which  are  also  indistinguishable  by 
inoculation  of  pigs.  The  only  difference  by  laboratory  experiment  which 
has  thus  far  been  brought  out  is  that  there  occur  Schweineseuche  bacilli  of 
higher  degree  of  virulence  as  tested  on  pigs  than  any  swine-plague  bac- 
teria which  have  hitherto  been  isolated  from  pigs  in  this  country.  Another 
point  to  be  considered  in  this  connection  is  that  Schweineseuche  occurs  as 
an  independent  disease  in  Germany  without  association  with  hog-  cholera, 
whereas  swine  plague  has  not  been  shown  to  prevail  with  the  same  inde- 
pendence as  an  epizootic  in  this  country.'' 

Silberschmidt  (1895)  arrives  at  a  different  conclusion  from  that  reached 
by  Smith,  Welch,  Bang,  and  others,  He  believes  that  the  diseases  of  swine 
known  as  hog  cholera,  swine  plague,  and  infectious  pneumo-enteritis  are  all 
due  to  one  and  the  same  bacillus,  which,  however,  varies  considerably  both 
in  its  morphological  characters  and  its  pathogenic  power.  In  view  of  the 
results  previously  reached  by  equally  competent  bacteriologists,  and  especially 
by  Smith  and  by  Welch  in  this  country,  we  are  not  disposed  to  accept  the 
view  maintained  by  Silberschmidt. 

Smith  has  described  several  varieties  of  the  hog-cholera  bacillus,  and  in 
his  account  of  the  "hog-cholera  group  of  bacteria "  shows  that  the  Bacillus 
enteriditis  of  Gartner  and  the  Bacillus  typhi  murium  of  Loffler  belong  to 
this  group.  The  characters  of  the  different  varieties  (or  species?)  belonging 
to  the  group  are  given  by  Smith  in  detail  (United  States  Department  of  Agri- 
culture, Bureau  of  Animal  Industry,  Bulletin  No.  6,  1894),  and  the  follow- 
ing1 general  statement  is  made: 

"  If  we  attempt  to  sum  up  those  characters  which  are  to  circumscribe  the 
hog-cholera  group  of  bacteria  we  are  at  once  confronted  by  the  scarcity  of 
common  characters.  Pathogenesis,  though  of  great  importance  from  the 
standpoint  of  pathology,  is  probably  the  last  character  acquired  and 
evidently  the  most  variable  and  most  readily  lost.  If  we  base  the  unity 
of  this  group  on  morphological  and  biological  characters,  we  are  like- 
wise met  by  variations  in  size,  absence  of  motility,  variations  in  the  ap- 
pearance of  the  colonies.  There  are,  however,  certain  underlying  char- 
acters, as  expressed  by  the  behavior  of  these  bacteria  in  bouillon  con- 
taining dextrose,  saccharose,  and  lactose,  which  I  think  will  serve  as  a  very 
important  group  character,  differentiating  such  groups  sharply  from  the 
colon  group.  I  would  therefore  suggest  that  for  the  present  all  bacteria 
whose  size  approximates  that  of  this  group,  which  do  not  liquefy  gelatin,  and 
whose  fermentative  properties  are  the  same  as  those  described  for  this  group, 
should  be  ranged  under  it.  Future  investigations  into  the  biochemical  char- 
acters of  these  varieties  or  sub-species  may  reveal  other  differential  charac- 
ters, but  the  time  has  not  yet  come  when  such  laborious  work  will  be  under- 
taken oi»  a  sufficiently  extensive  scale  to  be  of  any  service  in  differentiating 
varieties  and  sub-species." 

Selander  in  1890,  and  Metschnikoff  in  1892,  have  reported  a  rapid  increase 
in  virulence  of  the  bacillus  of  hog  cholera  by  successive  inoculations  in 
rabbits  or  pigeons.  Moore  (1894)  has  shown  that  this  is  a  mistake,  and  that 
the  bacteriologists  named  probably  did  not  experiment  with  cultures  of  the 
hog-cholera  bacillus,  as  they  supposed,  but  that  their  experiments  were 
made  with  the  bacillus  of  swine  plague — Bacillus  septicaemias  hemorrhagi- 
cae — which  when  passed  through  a  series  of  rabbits  attains  a  notable  increase 
in  pathogenic  virulence. 

In  a  recent  article,  Klein,  of  London  (1895)  says:  "  The  bacillus  of 
English  swine  plague,  which  I  described  in  1884,  in  Virchow's  Archiv,  as 


508 

shown  by  Smith  and  Welch,  is  identical  with  the  bacillus  of  American  hog 
cholera." 


BACILLUS   OF   BELFANTI   AND    PASCAROLA. 

Synonym. — Impftetanusbacillus. 

Obtained  by  Belfanti  and  Pascarola  (1888)  from  the  pus  of  wounds  in  an 
individual  who  succumbed  to  tetanus. 

Morphology. — Bacilli  with  rounded  ends,  sometimes  so  short  as  to  resemble 
micrococci;  resemble  the  Bacillus  septicaemias  haemorrhagicae  (fowl  cholera). 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method.  The 
ends  are  commonly  more  deeply  stained  than  the  central  portion. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  yel- 
lowish-gray, finely  granular,  spherical  colonies  with  smooth  outlines  are 
developed.  In  gelatin  stab  cultures,  at  18°  to  25°  C.,  at  the  end  of  twenty- 
four  hours  small,  spherical  colonies  are  developed  along1  the  line  of  punc- 
ture, which  are  isolated  or  closely  crowded;  upon  the  surface  a  rather  thin, 
shining,  grayish- white,  iridescent,  circular  layer  is  formed ;  gas  is  given  off 
which  has  not  a  disagreeable  odor.  Upon  the  surface  of  agar  elevated, 
shining,  gray  colonies  develop  along  the  impfstrich,  or  a  gray,  shining  band 
is  formed  which  increases  in  thickness  but  not  in  breadth — usually  less  than 
one-half  centimetre  broad.  Old  cultures  give  off  an  acid  odor.  Upon  blood 
serum  a  thin,  white  layer  is  developed  along  the  line  of  inoculation.  Upon 
potato  a  thin,  white,  varnish-like  layer  is  formed. 

Pathogenesis. — Very  pathogenic  for  rabbits,  guinea-pigs,  white  mice,  and 
sparrows.  Not  pathogenic  for  chickens,  pigeons,  or  geese. 


BACILLUS   OF   SWINE   PLAGUE,    MARSEILLES. 

Synonyms. — Bacillus  der  Schweineseuche,  Marseilles  (Rietsch 
and  Jobert)  ;  Bacillus  der  Frettchenseuche — ferret  disease  (Eberth 
and  Schimmelbusch) ;  Bacillus  der  Amerikanischen  Kinderseuche 
(Caneva) ;  Bacillus  of  spontaneous  rabbit  septicaemia  (Eberth). 

The  researches  of  Caneva  and  of  Bunzl-Federn  agree  as  to  the 
identity  of  the  bacillus  obtained  by  Rietsch  and  Jobert  (1887)  from 
swine  attacked  with  a  fatal  epidemic  disease  in  Marseilles,  and  the 
bacillus  found  by  Eberth  and  Schimmelbusch  (1889)  in  the  blood  of 
ferrets  suffering  from  a  fatal  form  of  septicaBmia  studied  by  them. 
The  first-named  bacteriologist  also  identifies  a  bacillus  supposed 
by  Billings  to  be  the  cause  of  "  Texas  fever  "  in  cattle  ("  Ameri- 
kanische  Rinderseuche  ")  and  the  bacillus  of  swine  plague  (Billings) 
with  the  above.  Bunzl-Federn  obtained  cultures  of  Billings7  swine- 
plague  bacillus  at  two  different  times.  He  identifies  the  one  first  re- 
ceived with  the  bacillus  now  under  consideration,  and  the  other  with 
the  bacillus  of  hog  cholera  (Salmon).1 

1  The  author  named  says:  '-With  reference  to  the  bacillus  of  swine  plague 
(Billings),  I  obtained,  as  did  Caneva,  a  decided  production  of  acid  in  the  cultures 
first  sent  by  Billings  ;  but  upon  testing  later  cultures  received  directly  from  Bil- 


IX   SUSCEPTIBLE   ANIMALS.  509 

Morphology. — Bacilli  with  rounded  ends,  about  twice  as  long  as 
broad,  and  one-third  smaller  than  the  bacillus  of  typhoid  fever 
(Eberth  and  Schimmelbusch).  The  bacillus  of  hog  cholera  is  shorter 
and  more  slender  than  the  Marseilles  bacillus,  and  the  bacillus  of 
Loffler  and  Schiitz  is  still  smaller  (Rietsch  and  Jobert). 

In  stained  preparations  the  extremities  of  the  rods  are  usually 
deeply  stained,  while  the  central  portion  remains  unstained—  :i  polar 
staining. "  By  Loffler's  method  of  staining  the  presence  of  flagella 
may  be  demonstrated  (Frosch). 

Stains  readily  with  the  aniline  dyes  usually  employed,  but  does 
not  retain  its  color  When  treated  by  Gram's  method. 

Biological  Characters. — An  aerobic  (facultative  anaerobic), 
non-liquefying,  actively  motile  bacillus.  Grows  readily  at  the 
room  temperature,  and  is  distinguished  from  the  bacillus  of  septi- 
caemia hsemorrhagica  by  its  active  movements  and  more  rapid  and 
abundant  development  in  the  various  culture  media  usually  em- 
ployed. It  is  distinguished  from  the  bacillus  of  hog  cholera  by  pro- 
ducing phenol  and  indol  in  solutions  containing  peptone,  by  causing 
coagulation  of  milk,  and  by  producing  an  acid  reaction  in  this  fluid. 
Grows  in  culture  media  having  an  acid  reaction. 

Rietsch  and  Jobert  give  the  following  account  of  the  characters 
of  growth  in  various  culture  media,  as  compared  with  the  bacillus  of 
hog  cholera  and  the  bacillus  of  Schweineseuche  (Loffler,  Schutz)  : 

Gelatin  streak  cultures.  At  the  end  of  twenty-four  hours  this 
bacillus  had  developed  considerably,  while  the  growth  of  the  hog- 
cholera  bacillus  was  scarcely  to  be  discerned  with  the  naked  eye,  and 
the  bacillus  of  Schweineseuche  did  not  form  a  visible  growth  until 
the  end  of  forty-eight  hours.  After  several  days  the  bacillus  of 
swine  plague  (Marseilles)  formed  an  opaque,  yellowish- white  streak, 
which,  when  examined  with  a  low-power  lens,  had  a  brown  color  by 
transmitted  light  and  a  bluish- white  color  by  reflected  light.  The 
streak  of  the  Loffler-Schiitz  bacillus  was  not  so  thick  and  not  so 
opaque,  and  was  made  up  of  small,  nearly  transparent  colonies  ;  the 
hog-cholera  bacillus  came  between  the  other  two.  Upon  blood 
serum,  agar,  and  glycerin-agar  the  Marseilles  bacillus  grew  more 
rapidly  than  the  other  two,  forming  a  layer  which  was  opaque  and 
of  a  white  color,  with  bluish  and  reddish  reflections.  Upon  potato 
it  formed  a  thick,  opaque,  yellowish  layer,  while  the  growth  of  the 
hog-cholera  bacillus  was  much  thinner  and  that  of  the  Loffler-Schiitz 
bacillus  scarcely  to  be  seen.  In  bouillon  the  Loffler-Schiitz  bacillus, 
at  the  end  of  three  days  at  37°  C.,  had  not  produced  any  perceptible 

lings  and  from  other  sources,  the  result  was  exactly  the  opposite — viz.,  a  decided 
production  of  alkali  in  milk  and  identity  with  the  hog-cholera  bacillus  of  Salmon." 


510  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

cloudiness,  while  the  Marseilles  bacillus  at  the  end  of  twenty-four 
hours  had  caused  the  fluid  to  be  clouded,  a  film  of  bacteria  had 
formed  upon  the  surface  and  a  deposit  at  the  bottom  of  the  tube  ;  the 
hog-cholera  bacillus  produced  a  less  degree  of  opacity  in  the  bouillon. 

Pathogenesis. — This  bacillus  is  pathogenic  for  sparrows  and 
other  small  birds  when  injected  beneath  the  skin  in  small  amounts, 
and  also  for  pigeons  in  a  longer  time — five  to  fourteen  days.  Frosch 
reports  a  negative  result  from  subcutaneous  injections  into  rabbits, 
guinea-pigs,  mice,  and  pigeons,  but  his  cultures  appear  to  have  be- 
come attenuated,  as  the  recent  cultures  of  Eberth  and  Schimmelbusch 
were  fatal  to  pigeons  in  four  out  of  five  experiments.  Two  rabbits 
were  inoculated  subcutaneously  by  Rietsch  and  Jobert  with  half  a 
Pravaz  syringef  ul  of  a  pure  culture  of  the  Marseilles  bacillus  ;  one  of 
these  died  on  the  sixth  day  and  the  other  survived. 

In  sparrows,  which  succumb  in  from  twenty-four  to  thirty-six 
hours  after  receiving  a  small  amount  of  a  pure  culture  in  the  breast 
muscle,  the  bacillus  is  present  in  the  blood  in  large  numbers,  and  a 
purulent  pleuritis  and  pericarditis  is  found  at  the  autopsy.  In  the 
ferrets  from  which  Eberth  and  Schimmelbusch  obtained  their  cultures 
the  bacillus  was  not  present  in  the  blood  in  sufficient  numbers  to  be 
readily  demonstrated  by  microscopical  examination,  but  it  was  ob- 
tained in  pure  cultures  from  the  liver,  spleen,  and  lungs.  The  prin- 
cipal pathological  appearances  noted  were  enlargement  of  the  spleen 
and  pneumonia.  Caneva  reports  that  the  Marseilles  bacillus  injected 
into  white  mice  gives  rise  to  an  extensive  abscess  at  the  point  of  in- 
oculation, but  does  not  kill  adult  animals.  In  a  young  mouse  which 
succumbed  to  such  an  injection  the  bacilli  were  not  generally  dis- 
tributed in  the  tissues,  but  were  found  as  emboli  in  the  smaller  capil- 
laries. This  bacillus,  then,  is  distinguished  from  the  similar  bacilli 
previously  described  by  its  comparatively  slight  pathogenic  power, 
as  well  as  by  its  more  vigorous  growth  in  culture  media,  and  the 
other  characters  heretofore  mentioned. 

BACILLUS    SEPTICUS   AGRIGENUS. 

Obtained  by  Nicolaier  from  soil  which  had  been  manured. 

Morphology. — Resembles  the  bacillus  of  fowl  cholera  and  of  rabbit  sep- 
ticaemia, of  which  it  is  perhaps  a  variety,  but  is  usually  somewhat  longer. 
It  also  sometimes  shows  the  end-staining  characteristic  of  Bacillus  septicae- 
mias haemorrhagicae,  but  not  so  constantly  and  not  so  sharply  defined. 

Biological  Characters. — An  aerobic,  (non-liquefying  ?),  non- motile  ba- 
cillus. Does  not  form  spores. 

In  gelatin  plate  cultures  spherical,  finely  granular  colonies  are  developed 
having  a  yellowish-brown  central  portion,  which  is  separated  by  a  dark 
ring  from  a  grayish-brown  marginal  zone;  later  this  difference  in  color  dis- 
appears and  the  colonies  become  more  decidedly  granular.  In  stick  cultures 
the  growth  consists  of  a  thin  layer  which  is  not  at  all  characteristic. 

Pathogenesis. — Small  quantities  of  a  pure  culture  injected  into  the  ear 


IX   SUSCEPTIBLE   ANIMALS.  511 

vein  of  a  rabbit  cause  its  death  in  from  twenty-four  to  thirty-six  hours; 
pathogenic  also  for  house  mice  and  for  field  mice.  At  the  autopsy  no  notable 
pathological  changes  are  observed.  The  bacilli  are  found  in  blood  from  the 
heart  and  in  the  capillaries  of  the  various  organs,  but  are  not  so  numerous 
as  in  rabbit  septicaBmia;  they  show  a  special  inclination  to  adhere  to  the 
margins  of  the  red  blood  corpuscles. 


BACILLUS   ERYSIPELATOS   SUIS. 

Synonyms. — Bacillus  of  hog  erysipelas;  Bacillus  des  Schweine- 
rothlauf  (Loffler,  Schutz)  ;  Bacille  du  rouget  du  pore  (Pasteur)  ;  Ba- 
cillus of  mouse  septicaemia;  Bacillus  murisepticus  (Fliigge)  ;  Bacil- 
lus des  Mauseseptikamie  (Koch). 

The  bacillus  of  mouse  septicaemia,  first  described  by  Koch  (1878), 
resembles  so  closely  in  its  morphology,  characters  of  growth,  and 
pathogenic  power  the  bacillus  of  Schweinerothlauf  of  Loffler  and 
Schutz  (1885)  that  they  can  scarcely  be  considered  as  distinct  spe- 
cies, although,  from  slight  differences  which  have  been  observed,  they 
are  perhaps  entitled  to  separate  consideration  as  varieties  of  the 
same  species.  Fliigge,  Eisenberg,  Frankel,  and  other  authors,  while 
recognizing  the  fact  that  the  bacilli  from  the  two  sources  closely  re- 
semble each  other,  apparently  do  not  consider 
them  identical,  and  describe  them  separately. 
Baumgarten,  on  the  other  hand,  describes  them 
under  one  heading  and  considers  it  highly  prob- 
able that  they  are  identical,  although  he  also 
admits  slight  differences  in  the  morphological 
characters  and  growth  in  culture  media.  These 
differences  are,  however,  no  greater  than  we 
have  in  artificially  produced  varieties  of  other  FlG  133_Baclllus  of 
well-known  microorganisms,  and  we  think  it  mouse  septicaemia  in  leu- 
best  to  follow  Baumgarten  in  describing  them 
under  a  single  heading. 

Koch  first  obtained  this  bacillus  by  injecting  putrefying  blood  or 
flesh  infusion,  during  the  first  days  of  putrefactive  change,  beneath 
the  skin  of  mice.  A  certain  proportion  of  the  animals  experimented 
upon  contracted  a  fatal  form  of  septicaemia,  and  the  bacillus  under 
consideration  was  found  in  their  blood.  The  bacillus  of  Schweine- 
rothlauf was  obtained  by  Loffler  and  by  Schutz  from  the  blood  and 
various  organs  of  swine  which  had  succumbed  to  the  infectious 
malady  known  in  Germany  as  rothlauf  and  in  France  as  rouget. 

Morphology. — Extremely  minute  bacilli,  about  1  v  in  length  and 
0.2  v  in  diameter.  The  Schweinerothlauf  bacilli  are  described  as 
somewhat  thicker  and  longer  by  Fliigge,  by  Frankel,  and  by  Ei- 
senberg, but  Baumgarten  states  that  they  are  somewhat  more 


512 


BACILLI    WHICH   PRODUCE   SEPTIC^MIA 


slender  and  on  the  average  shorter  than  the  bacillus  of  mouse  septi- 
ca3mia.  The  bacilli  are  solitary,  or  in  pairs  the  elements  of  which 
are  often  united  at  an  angle  ;  occasionally  a  chain  of  three  or  four 
elements  may  be  observed,  and  in  old  cultures  the  bacilli  may 
grow  out  into  short  threads  which  are  straight  or  more  or  less 
curved  and  twisted.  Small  refractive  bodies  may  sometimes  be  dis- 
tinguished in  the  rods,  and  these  have  been  supposed  by  some  authors 
to  be  spores,  but  this  has  not  been  demonstrated. 

This  bacillus  stains  readily  with  the  ordinary  aniline  staining 
agents  and  also  by  Gram's  method. 

Biological  Characters. — A. facultative  anaerobic,  non-liquefy- 
ing bacillus.  According  to  Schottelius,  the  rothlauf  bacilli  are  some- 


FIG.  134.— Bacillus  of  rouget,  from  a  pure  culture.     X  1,000.    From  a  photomicrograph.     (Roux.) 

times  motile,  but  Fliigge  states  that  other  observers  have  not  seen 
them  in  active  motion.  Frankel  says  they  have  the  power  of  volun- 
tary motion.  Eisenberg  says  that  the  bacillus  of  mouse  septica3mia 
is  motionless,  and  Frankel  says  they  "  seem  to  be  incapable  of  volun- 
tary motion. "  Baumgarten  remarks  :  "  Whether  the  bacilli  exhibit 
voluntary  movements  has  not  been  determined."  Although  this 
bacillus  is  not  strictly  anaerobic,  it  grows  better  in  the  absence  of 
oxygen  than  in  its  presence.  Development  occurs  in  various  culture 
media  at  the  room  temperature,  but  is  more  rapid  in  the  culture 
oven.  In  gelatin  stab  cultures  no  development  occurs  upon  the 
surface,  but  the  growth  along  the  line  of  puncture  is  very  character- 
istic; this  consists  of  a  delicate  cloud-like,  radiating  growth,  which 
extends,  in  the  course  of  a  few  days,  almost  to  the  walls  of  the  test 
tube.  The  rothlauf  bacillus  does  not  extend  so  rapidly  through  the 


iN   SUSCEPTIBLE   ANIMALS. 


513 


gelatin,  and  the  branching,   cloud-like  growth  is  not   as  delicate; 

Flugge  compares  it  to  the  brush  of  bristles  used  for  cleansing  test 

tubes.     In    old    cultures    in  nutrient   gelatin    a 

slight  softening  of  the  gelatin  occurs  along  the 

line  of  growth,  and  as  a  result  of  evaporation 

and  desiccation  a  funnel-shaped  cavity  is  formed 

in  the  culture  medium  in  the  course  of  two  or 

three  weeks.      In  gelatin  plates   colonies    are 

developed  in  the  course  of  two  or  three  days  in 

the  deeper  layers  of  the  gelatin,   but  not  upon 

the   surface;    these   are  nebulous,  grayish-blue, 

radiating  masses,  which  are  so  delicate  as  to  be 

scarcely  visible  without  the  aid  of  a  lens  or  a 

dark    background.      Under  a  low   power  they 

appear   as    branching   feathery    masses,    which 

have  been  compared  by  Flugge  to  the  radiating 

growth  of  "  bone  corpuscles."     In  older  cultures 

they  coalesce  and  cause  a  nebulous  opacity  of 

the  whole  plate,  which  has  a  bluish-gray  lustre. 

Upon  the  surface  of  nutrient  agar  or  blood 
serum  a  very  scanty  development  occurs  along 
the  line  of  inoculation.  No  growth  occurs  upon 
potato.  In  bouillon  the  bacilli  cause  a  slight 
cloudiness  at  the  outset,  and  later  a  scanty  gray- 
ish-white deposit  upon  the  bottom  of  the  test 
tube ;  no  film  is  formed  upon  the  surface. 

The  thermal  death-point  of  this  bacillus,  as  determined  by  the 
writer  (1887),  is  58°  C.,  the  time  of  exposure  being  ten  minutes.  In 
the  experiments  of  Bolton  it  was  destroyed  in  two  hours  by  mercuric 
chloride  solution  in  the  proportion  of  1 : 10,000;  by  carbolic  acid  and 


FIG.  135.  —  Bacillus  of 
mouse  septicaemia; 
culture  in  nutrient  gela- 
tin, end  of  four  days  at 
18°  C.  (Baumgarten.) 


FIG.  13H.— Bacillus  of  mouse  septicaemia;  single  colony  in  nutrient  gelatin.    X  80.    (Flugge.) 

by  sulphate  of  copper  in  one-per-cent  solution.     These  results  are 
opposed  to  the  view  that  the  minute  refractive  granules  which  may 
sometimes  be  seen  in  the  interior  of  the  rods  are  reproductive  spores, 
33 


514 


BACILLI  WHICH   PRODUCE   SEPTIC^MIA 


for  all  known  spores  have  a  much  greater  resisting  power  to  heat 
and  the  chemical  agents  named. 

Pathogenesis.— Pathogenic  for  swine,  rabbits,  white  mice,  house 
mice,  pigeons,  and  sparrows.  Field  mice,  guinea-pigs,  and  chickens 
are  immune. 

Swine  may  be  infected  by  the  ingestion  of  food  containing  the 
rothlauf  bacillus,  as  has  been  demonstrated  by  allowing  them  to  eat 
the  intestine  of  an  animal  which  had  recently  succumbed  to  the  dis- 
ease, and  also  by  the  subcutaneous  injection  of  pure  cultures.  The 
disease  usually  terminates  fatally  within  three  or  four  days,  and 
sometimes  in  less  than  twenty-four  hours.  It  is  characterized  by 


FIG.  137.— Section  of  diaphragm  of  a  mouse  dead  from  mouse  septicaemia,  showing  bacilli  in  a 
capillary  bloodvessel.     (Baumgarten.) 

fever,  debility,  loss  of  appetite,  and  by  the  appearance  upon  the  sur- 
face of  the  body  of  red  patches,  which  gradually  extend  and  become 
confluent,  producing  after  a  time  a  uniform  dark-red  or  brown  color 
of  the  entire  surface.  The  discharges  from  the  bowels  frequently 
contain  bloody  mucus.  At  the  autopsy,  in  acute  cases,  the  spleen  is 
notably  enlarged,  and  the  liver  and  kidneys  are  likely  to  be  more  or 
less  swollen,  as  are  also  the  lymphatic  glands,  especially  those  of 
the  mesentery;  the  gastric  and  intestinal  mucous  membranes  are 
usually  inflamed  and  spotted  with  ha3morrhagic  extravasations  ;  the 
serous  membranes  also  may  be  inflamed,  and  the  cavities  of  the 
pleura3,  pericardium,  and  peritoneum  usually  contain  more  or  less 
fluid.  The  bacilli  are  found  in  the  blood  vessels  throughout  the 
body  and  are  especially  numerous  in  the  interior  of  the  leucocytes. 


PL  ATE  VI. 

STERNBERG'S  BACTERIOLOGY. 


.   i. 


Fig.   2. 


Figr.  3. 


Fig.  4. 


Fig.  6. 


IN  SUSCEPTIBLE  ANIMALS.  515 

Cornevin  and  Kitt  have  shown  that  the  contents  of  the  intestine 
also  contain  the  bacilli  in  large  numbers,  and  the  disease  appears  to 
be  propagated  among  swine  principally  by  the  contamination  of  their 
food  with  the  alvine  discharges  of  diseased  animals. 

Pigeons  are  very  susceptible  to  the  pathogenic  action  of  this  ba- 
cillus, and  usually  die  within  three  or  four  days  after  inoculation 
with  a  pure  culture.  Rabbits  are  not  so  susceptible,  although  a 
certain  proportion  die  from  general  infection  after  being  inoculated 
in  the  ear.  The  first  effect  of  such  an  inoculation  is  to  produce  an 
erysipelatous  inflammation.  When  the  animal  recovers  it  is  subse- 
quently immune. 

White  mice  and  house  mice  are  extremely  susceptible,  but  field 
mice  are  immune.  This  remarkable  fact  was  first  ascertained  by 
Koch  by  experiments  with  his  bacillus  of  mouse  septicaemia.  House 
mice  which  have  been  inoculated  with  a  minute  quantity  of  a  pure 
culture  of  the  rothlauf ,  or  mouse  septicaemia,  bacillus,  die  in  from 
forty  to  sixty  hours.  The  animal  is  usually  found  dead  in  a  sitting 
position,  with  its  back  strongly  curved,  and  for  many  hours  before 
death  it  remains  quietly  sitting  in  the  same  position  ;  the  eyes  are 
glued  together  by  a  sticky  secretion  from  the  conjunctival  mucous 
membrane.  At  the  autopsy  the  spleen  is  found  to  be  very  much  en- 
larged, and  there  may  be  a  slight  amount  of  oedema  at  the  point  of 
inoculation. 

The  bacilli  are  found  in  the  blood  vessels  generally,  and  are  very 
numerous  in  the  interior  of  the  leucocytes,  which  are  sometimes  com- 
pletely filled  with  them. 


BACILLUS  COPROGENES   PARVUS. 

Synonym. — Mauseseptikamieahnlicher  Bacillus  (Eisenberg). 

Obtained  by  Bienstock  from  human  faeces. 

Morphology.— A.  very  minute  bacillus,  which  is  but  little  longer  than  it 
is  broad,  and  might  easily  be  mistaken  for  a  micrococcus. 

Biological  Characters. — Grows  very  slowly  on  nutrient  gelatin,  forming 
a  scarcely  visible  film  along  the  line  of  inoculation,  which  at  the  end  of 
several  weeks  is  scarcely  one  millimetre  wide.  Is  not  motile. 

Pathogenesis. — In  white  mice  an  extensive  oedema  is  developed  at  «the 
point  of  inoculation  at  the  end  of  ten  or  twelve  hours,  and  the  animal  dies 
within  thirty- six  hours.  The  bacilli  are  found  in  great  numbers  in  the 
effused  serum  at  the  point  of  inoculation  and  in  comparatively  small  num- 
bers in  the  blood.  A  rabbit  inoculated  with  a  pure  culture  obtained  from  a 
mouse  died  at  the  end  of  eight  days.  The  inoculation,  which  was  made  in 
the  ear,  gave  rise  to  a  local  erysipelatous  inflammation. 


516 


BACILLI   WHICH   PRODUCE   SEPTICAEMIA 


BACILLUS   CAVICIDA. 

Synonym. — Brieger's  bacillus.  Probably  a  pathogenic  variety  of  Bac- 
terium coli  commune  of  Escherich. 

Obtained  by  Brieger  (1884)  from  human  faeces. 

Morphology. — Small  bacilli,  about  twice  as  long  as  broad,  which  closely 
resemble  the  colon  bacillus  of  Escherich  (Bacterium  coli  commune). 

Biological  Characters. — An  aerobic  (facultative  anaerobic),  non-liquefy- 
ing bacillus. 

The  growth  in  gelatin  plate  cultures  is  said  to  be  very  characteristic,  the 
colonies  being  "  in  the  form  of  very  beautifully  grouped,  whitish,  concentric 
rings,  which  are  arranged  like  the  ccales  upon  the  back  of  a  turtle  "  (Eisen- 
berg).  The  writer  has  studied  cultures  of  this  bacillus  brought  from  the 
bacteriological  laboratories  of  Germany,  side  by  side  with  cultures  of  the 
Bacterium  coli  commune  of  Escherich,  and  has  found  no  appreciable  differ- 
ences in  the  colonies  in  gelatin  plates,  or  in  the  growth  in  various  culture 
media.  Upon  potato  it  grows  rapidly  in  the  incubating  oven,  forming  a 
dirty-yellow,  moist  layer. 

Pathogenesis. — This  bacillus,  as  first  obtained  by  Brieger,  was  character- 
ized by  being  very  pathogenic  for  guinea-pigs, which  were  invariably  killed, 
within  seventy- two  hours,  by  the  subcutaneous  injection  of  a  minute  quan- 
tity of  a  pure  culture.  The  bacillus  was  found  in  great  numbers  in  the 
blood  of  animals  which  succumbed  to  an  experimental  inoculation.  The 
writer's  experiments  with  this  bacillus,  made  in  1889,  indicate  that  its  patho- 
genic power  had  become  attenuated,  inasmuch  as  considerable  quantities  of 
a  pure  culture  injected  into  guinea-pigs  did  not  cause  the  death  of  the  ani- 
mals— culture  used  came  originally  from  Germany.  Not  pathogenic  for 
rabbits  or  for  mice. 


BACILLUS   CAVICIDA   HAVANIENSIS. 

This  bacillus  was  obtained  by  the  writer  from  the  contents  of  the  intestine 
of  a  yellow-fever  cadaver,  in  Havana,  1889,  through  inoculated  guinea-pigs. 

Morphology. — A  bacillus  with  rounded  ends, 
from  two  to  three  u  long  and  about  0.7  p-  broad, 
frequently  united  in  pairs. 

Stains  readily  with  the  ordinary  aniline  colors. 
Biological  Characters. — An  aerobic  and  fac- 
ultative anaerobic,  non  -liquefying,  actively  mo- 
tile bacillus. 

In  gelatin  stab  cultures  the  growth  upon  the 
surface  is  very  scanty  and  thin,  not  extending  far 
from  the  point  of  puncture  ;  along  the  line  of 
puncture  are  developed  small,  translucent,  pearl- 
like,  spherical  colonies,  which  later  become  opaque 
and  sometimes  granular.  In  gelatin  roll  tubes, 
at  the  end  of  twenty- four  hours  at  22°  C., 
the  deep  colonies  are  very  small  spheres,  of  a  pale 
stra  w  color ;  later  they  become  opaque,  light-brown 
spheres,  or  may  have  a  dark  central  mass  sur- 
rounded by  a  transparent  zone.  The  superficial 

colonies  at  the  end  of  five  days  are  small,  translucent  masses  of  a  pale  straw 
color  towards  the  centre,  with  thin  and  irregular  margins,  sometimes  with 
a  central  light-brown  nucleus ;  at  the  end  of  ten  days  the  deep  colonies  are 
still  quite  small,  of  a  brown  color,  and  opaque. 

In  gtycerin-agar  roll  tubes,  at  the  end  of  twenty-four  hours,  the  deep  colo- 
nies are  in  the  form  of  a  biconvex  lens,  and  appear  spherical  when  viewed 
in  face  and  biconvex  when  seen  from  the  side ;  they  have  a  straw  color 


FIG.  138.— Bacillus  cavicida 
Havaniensis;  from  a  potato 
culture.  X  1,000.  From  a  pho- 
tomicrograph. (Sternberg.) 


IN  SUSCEPTIBLE  ANIMALS, 


517 


by  transmitted  light  and  are  bluish-white  by  reflected  light ;  the  superficial 
colonies  are  translucent,  with  a  bluish- white  lustre. 

On  potato,  at  22°  C.,  at  the  end  of  forty  eight  hours  there  is  a  thin,  dirty- 
yellow  growth  of  limited  extent;  at  the  end  of  ten  days  there  is  a  thin, 
gamboge  yellow  layer  and  little  masses  of  the  same  color;  the  growth  is 
quite  thin,  with  irregular  outlines,  and  is  confined  to  the  vicinity  of  the 
impfstrich. 

Grows  in  nutrient  agar  containing  0.2  per  cent  of  hydrochloric  acid. 
Thermal  death  point  55°  C.  Grows  in  agua  coco  without  forming  gas,  and 
causes  this  liquid  and  bouillon  to  become  slightly  translucent — not  milky. 

Pathogenesis. — Pathogenic  for  guinea-pigs,  less  so  for  rabbits.  Guinea- 
pigs  inoculated  subcutaneously  with  a  few  drops  of  a  pure  culture  die  in  ten 
or  twelve  hours  from  general  infection.  There  is  usually  a  considerable 
effusion  of  bloody  serum  in  the  vicinity  of  the  point  of  inoculation,  and  the 
spleen  is  more  or  less  enlarged. 

BACILLUS   CRASSUS   SPUTIGENUS. 

Obtained  by  Kreibohm  (1886)  from  the  sputum  of  two  individuals,  and 
once  in  scrapings  from  the  tongue. 

Morphology. — Short,  thick  bacilli,  of  oblong  form,  with  rounded  corners, 
often  bent  or  twisted — "sausage-shaped."  Immediately  after  division  the 
bacilli  are  about  one-half  longer  than  they  are  broad,  but  before  dividing 


^m^ymm 

f£^/-:  &:?%*'.  : --s^^cs  •"';  -Kr<^^^^p:;%^^v':' 
;  ._  :       •  ^ffiSSS^^^Jri^- 


FIG.  139.—  Bacillus  crassus  sputigenus,  from  blood  of  mouse,    x  700.    (Flugge.) 


again  they  may  attain  a  length  of  three  to  four  times  the  breadth.  Irregular 
forms  with  swollen  ends  or  uneven  contour  are  frequently  seen. 

This  bacillus  is  quickly  stained  by  the  ordinary  aniline  colors  and  also 
by  Gram's  method. 

Biological  Characters.  —  An  aerobic,  non-liquefying  (non-motile  ?)  ba- 
cillus. Grows  in  various  culture  media  at  the  room  temperature  —  more 
rapidly  in  the  incubating  oven.  "  Appears  to  form  spores  at  35°  C." 
(Fliigge;. 

In  gelatin  plates,  at  the  end  of  thirty-six  hours,  grayish-  white  colonies  are 
developed,  which  soon  reach  the  surface  of  the  gelatin  and  spread  out  as 
round,  viscid,  grayish  white  drops,  which  project  considerably  above  the 
surface  of  the  culture  medium.  Under  a  low  magnifying  power  recent  colo- 


518  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

nies  appear  as  spherical,  grayish-brown  discs,  the  surface  of  which  is  marked 
with  dark  points  or  lines.  The  superficial  colonies  are  more  transparent, 
have  irregular  outlines,  and  the  surface,  especially  near  the  margins,  is 
coarsely  granular.  The  development  in  stab  cultures  is  very  rapid  and  re- 
sembles that  of  Friedlander's  bacillus — ' '  nail-shaped  "  growth.  Upon  potato 
the  growth  is  also  similar  to  that  of  Friedlander's  bacillus,  and  consists  of  a 
thick,  grayish- white,  moist,  and  shining  layer. 

Pathogenesis. — Mice  inoculated  with  a  small  quantity  of  a  pure  culture 
die  from  acute  septicaemia  in  about  forty-ei^ht  hours.  The  bacilli  are  found 
in  blood  from  the  heart  and  from  the  various  organs — most  numerous  in 
the  liver.  Eabbits  are  killed  within  forty  eight  hours  by  intravenous  injec- 
tion of  a  small  quantity,  and  the  blood  contains  the  bacillus  in  great  num- 
bers. Larger  amounts  injected  into  the  circulation  of  rabbits  or  dogs  cause 
death  in  a  few  hours  (three  to  ten),  preceded  by  diarrhoea,  and  in  some  in- 
stances bloody  discharges  from  the  bowels.  At  the  autopsy  an  acute  gastro- 
enteritis is  found. 


BACILLUS   PYOGENES   FCETIDUS. 

Obtained  by  Passet  (1885)  from  an  abscess  of  the  anus. 

Morphology.—  Short  bacilli  with  rounded  ends,  1.45  n  long  and  0.58  /u 
broad ;  usually  associated  in  pairs  or  in  short  chains. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Grows  rapidly  in  the  usual  culture  media  at  the  room  temperature.  In  the 
interior  of  the  rods,  in  stained  preparations,  one  or  two  unstained,  spherical 
places  may  sometimes  be  seen,  which  have  been  supposed  to  be  spores  (?). 
The  independent  motion  exhibited  by  this  bacillus  is  not  very  active.  In 
gelatin  plates  white  colonies  are  developed  at  the  end  of  twenty-four  hours, 
which  upon  the  surface  spread  out  as  grayish-white  plaques,  having  a  dia- 
meter sometimes  of  one  centimetre ;  these  are  thickest  in  the  centre  and  of 
a  whitish  color ;  the  colonies  may  become  confluent.  In  gelatin  stab  cul- 
tures the  growth  upon  the  surface,  at  the  end  of  twenty-four  hours,  consists 
of  a  thin,  grayish  white  layer  with  rather  thick,  irregular  margins ;  along  the 
line  of  puncture  more  or  less  crowded  colonies.  Upon  potato  ih&  bacillus 
forms  an  abundant,  shining,  pale-brown  layer.  The  cultures  give  off  a  dis- 
agreeable putrefactive  odor. 

According  to  Eisenberg,  mice  and  guinea-pigs  are  killed  in  twenty-four 
hours  by  injections  beneath  the  skin  or  into  the  cavity  of  the  abdomen,  and 
numerous  bacilli  are  found  in  the  blood. 


PROTEUS  HOMINIS  CAPSULATUS. 

Obtained  by  Bordoni-Uffreduzzi  (1887)  from  two  cadavers  presenting  the 
pathological  appearances  of  the  so-called  "  Haderiikrankheit. " 

Morphology. — Bacilli,  varying  considerably  in  dimensions;  somewhat 
thicker  than  the  anthrax  bacillus;  often  swollen  in  the  middle  or  at  the  ex- 
tremities; more  or  less  curved;  isolated,  united  in  pairs  or  in  long  filaments; 
in  stained  preparations  from  agar  cultures  or  from  blood  the  bacilli  are  sur- 
rounded by  a  "capsule." 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  (facultative  anaerobic  ?),  non-lique- 
fying, non  motile  bacillus.  Formation  of  spores  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  At  a  temperature  of  15°  to 
17D  C.  long  filaments  are  formed,  in  which  the  bacilli  are  surrounded  with  a 
capswle ;  at  22°  to  24°  C.  the  bacilli  are  for  the  most  part  isolated,  but  few  fila- 
ments being  formed  ;  at  32°  to  37°  C.  the  bacilli  are  so  short  as  to  resemble 
micrococci ;  development  ceases  at  a  temperature  of  8°  and  is  very  slow  at 
15°  C. 


IN   SUSCEPTIBLE   ANIMALS. 


519 


This  bacillus  grows  as  well  in  an  acid  medium  as  in  one  which  is  slightly 
alkaline.  In  gelatin  plates,  at  the  end  of  eighteen  to  twenty-four  hours, 
colonies  are  formed  which  under  a  low  power  are  seen  to  be  spherical  and 
to  contain  a  quantity  of  shining  granules ;  the  following  day,  at  a  tempera- 
ture of  15°  to  17°  C.,  the  colonies  may  be  as  large  as  a  pin's  head  and  still 
remain  spnerical  or  slightly  oval,  but 
the  outline  is  no  longer  so  uniform, 
and  between  the  shining  points  in  the 
interior  a  confused  network  may  be 
seen ;  as  the  colony  becomes  larger  it 
is  raised  above  the  surface  of  the  gela- 
tin, becomes  opaque,  and  has  a  pearly 
lustre  like  that  of  Friedlander's  bacil- 
lus. In  gelatin  stab  cultures  the 
growth  resembles  that  of  Friedlan- 
der's bacillus — "  nail-shaped  growth." 
Upon  the  surface  of  nutrient  agar  a 
rapidly  extending,  semi  transparent 
layer  is  formed.  Upon  potato,  at  15° ' 
to  17°  C.,  at  the  end  of  twenty-four 
hours  transparent  drops  are  seen  in 
the  vicinity  of  the  point  of  inocala- 
tion,  and  later  a  moist,  shining,  color- 
less layer,  of  tough  consistence,  is 
formed,  which  gradually  extends  over 


the  surface.  The  growth  upon  blood 
serum  resembles  that  upon  nutrient 
agar,  and  the  blood  serum  is  not  liquefied. 


Pia.  140.— Proteus  hominis  capsulatus,  from 
liver  of  mouse.  X  1,000.  (Bordoni-Uffre^ 
duzzi.) 


IMSL t»i ,  K/IJ.VI.   vi*.\j  ^ n.*~r-~r-~~  -^-^.^  «. *-^.  .»  — — „  ~ . ^ ~. .        i  liquid  Diood  serum  or  in. 

bouillon  the  bacilli  are  isolated — not  in  filaments;  they  cause  a  clouding  of 
the  liquid,  and  an  abundant  deposit  accumulates  at  the  bottom  of  the  tube, 
while  a  film  of  bacilli  forms  upon  the  surface.  The  cultures  never  give  off 
a  putrefactive  odor. 

Pathogenesis. — Pathogenic  for  dogs  and  for  mice,  less  so  for  rabbits  and 
for  guinea-pigs.  Agar  cultures  grown  in  the  incubating  oven  at  32°  to  37 
G.  are  more  pathogenic  than  cultures  in  gelatin  at  the  room  temperature. 
A  small  quantity  of  a  recent  culture  injected  subcutaneously  in  mice  cause* 
their  death  in  from  one  to  four  days,  according  to  the  quantity  and  age  of 
the  culture;  the  recent  cultures  are  most  virulent.  When  the  animal  lives 
more  than  twenty -four  hours  it  has  a  mucous  diarrhoea.  At  the  autopsy  the 
spleen  is  found  to  be  much  enlarged  and  dark  in  color  ;  the  lymphatic 
glands  are  also  swollen  and  haemorrhagic,  the  liver  and  kidneys  hyperaemic; 
in  the  vicinity  of  the  point  of  inoculation  is  a  subcutaneous  oadema  of  jelly- 
like  appearance  and  numerous  punctiform  haemorrhages  are  seen.  The  ba- 
cillus is  found  in  great  numbers  in  the  effused  serum  from  the  subcutaneous 
tissues,  in  the  blood,  the  contents  of  the  intestine,  and  in  the  parenchyma  of 
the  various  organs.  When  examined  at  once  the  bacilli  in  the  subcutaneous 
oedema  and  in  the  lymphatic  glands  are  usually  quite  short,  and  even  spherical, 
while  in  the  blood  they  are  somewhat  longer  and  may  appear  as  short  fila- 
ments with  swollen  ends,  surrounded  by  a  capsule.  When  the  examination 
is  made  some  time  after  the  death  of  the  animal  longer  filaments  are  quite 
numerous.  Rabbits  and  guinea-pigs  are  killed  by  the  intravenous  injection 
of  comparatively  small  amounts  of  a  recent  culture,  but  quite  large  doses 
are  required  to  produce  a  fatal  result  when  the  injection  is  made  beneath 
the  skin.  From  two  to  three  cubic  centimetres  of  a  recent  culture  injected 
into  the  circulation  of  a  dog  give  rise  to  symptoms  of  toxaemia,  and  the  ani- 
mal usually  dies  on  the  second  day.  At  the  autopsy  the  abdominal  organs 
are  found  to  be  hyperaemic,  the  mucous  membrane  of  the  intestine  swollen, 
red  in  color,  and  covered  with  bloody  mucus.  The  bacillus  is  found  in  the 
blood  and  in  the  various  organs.  When  smaller  doses  are  injected  into  a 
vein  (a  few  drops)  the  animal,  after  a  few  hours,  has  a  mucous  diarrhoea  and 


520  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

vomiting,  or  efforts  to  vomit.  Death  usually  occurs  at  the  end  of  two  or 
three  days.  At  the  autopsy  the  spleen  is  found  to  be  normal,  the  other  or- 
gans slightly  hyperaamic,  and  the  intestinal  mucous  membrane  in  a  state  of 
catarrhal  inflammation.  The  bacilli  are  found  in  the  blood  and  in  the  vari- 
ous organs  in  considerable  numbers. 

PROTEUS   CAPSULATUS   SEPTICUS. 

Obtained  by  Banti  (1888)  from  a  case  of  "  acute  haemorrhagic  infection." 
According  to  Banti,  this  is  possibly  identical  with  the  preceding  species — 

Proteus  hominis  capsulatus — but  in  some  respects  more  nearly  resembles 

Friedlander's  bacillus. 

BACILLUS  ENTERITIDIS. 

Obtained  by  Gartner  (1888)  from  the  tissues  of  a  cow  which  was  killed  in 
consequence  of  an  attack  characterized  by  a  mucous  diarrhosa,  and  also  from 
the  spleen  of  a  man  who  died  twelve  hours  after  eating  the  flesh  of  this 
animal. 

Morphology. — Short  bacilli,  about  twice  as  long  as  broad,  frequently  united 
in  pairs;  chains  of  four  to  six  elements  are  sometimes  seen. 

Stains  with  the  usual  aniline  colors,  and  presents  the  peculiarity  of 
staining  deeply  at  one  end  while  the  remainder  of  the  rod  is  but  slightly 
stained.  When  two  bacilli  are  united  the  deeply  stained  ends  are  in  apposi- 
tion. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Spore  formation  not  determined.  Grows  in  the  usual  culture  media  at  the 
room  temperature.  Upon  gelatin  plates  pale-gray,  superficial  colonies  are 
formed  at  the  end  of  twenty-four  hours;  under  a  low  power  these  are  seen 
to  be  coarsely  granular  and  transparent ;  the  central  portion  usually  pre- 
sents a  greenish  color  ;  deep  colonies  are  spherical,  indistinctly  granular, 
and  of  a  brownish  color  ;  in  older  colonies  a  marginal  transparent  zone  is 
seen  which  appears  to  be  made  up  of  minute  fragments  of  glass  of  a  pale- 
brown  color.  In  gelatin  stab  cultures  but  slight  development  occurs  along 
the  line  of  puncture  ;  upon  the  surface  a  thick,  grayish- white  layer  is 
formed,  which  after  a  time  becomes  very  much  wrinkled.  Upon  the  surface 
of  agar,  at  37°  C.,  at  the  end  of  eighteen  to  twenty  hours  a  grayish- yellow 
layer  has  formed.  Upon  potato  a  moist,  shining,  yellowish-gray  layer  is 
developed.  The  growth  upon  blood  serum  is  rapid  in  the  form  of  a  gray 
layer  along  the  line  of  inoculation. 

Pathogenesis. — White  mice  and  house  mice  usually  die  in  from  one  to 
three  days  when  fed  with  a  pure  culture  of  this  bacillus.  Babbits  and  gui- 
nea-pigs die  in  from  two  to  five  days  from  subcutaneous  injections— less 
pathogenic  for  pigeons  and  canary  birds.  Dogs,  cats,  chickens,  and  sparrows 
are  immune.  A.  goat  died  in  twenty  hours  after  receiving  an  intravenous 
injection  of  two  cubic  centimetres  of  a  culture  in  blood  serum.  The  princi- 
pal pathological  appearance  consists  in  an  intense  inflammation  of  the  in- 
testinal mucous  membrane.  The  bacilli  are  found  in  blood  from  the  heart 
and  also  in  the  contents  of  the  stomach. 

BACILLUS   OF   GROUSE   DISEASE. 

Obtained  by  Klein  (1889)  from  the  lungs  and  liver  of  grouse  which  had 
succumbed  to  an  epidemic  disease. 

Morphology. — Bacilli  with  rounded  ends,  from  0.8  to  1.6/^long;  may 
also  be  seen  as  spherical  or  oval  cells  0.6  /j.  long  and  0.4  jn  thick;  solitary,  in 
pairs,  or  in  chains  of  three  to  four  elements. 

Stains  best  with  Weigert's  solution  of  methylene  blue  in  aniline  water. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motile  bacillus. 
Spore  formation  not  observed.  Grows  in  the  usual  culture  media  at  the 


IN   SUSCEPTIBLE   ANIMALS.  521 

room  temperature — better  in  the  incubating-  oven.  Upon  gelatin  plates,  at 
20°  C.,  at  the  end  of  twenty-four  hours  small,  angular,  transparent  scales 
may  bo  seen  upon  the  surface  with  a  low-power  lens;  at  the  end  of  three  or 
four  days  these  form  flat,  more  or  less  irregular,  shining,  gray  colonies,  with 
thin  and  of  ten  dentate  margins ;  these  colonies  may  become  confluent  and 
form  a  dry,  scaly  layer  which  by  reflected  light  has  a  peculiar,  fatty  lustre 
In  gelatin  stab  cultures  the  superficial  growth  is  in  the  form  of  a  trans- 
parent, dry,  grayish  layer  with  dentate  margins,  not  more  than  three  to  five 
millimetres  in  diameter.  Upon  agar,  at  36°  to  37°  C.,  a  thin,  whitish-gray, 
dry  layer  is  formed. 

Pathogenesis. — Pathogenic  for  mice,  for  guinea-pigs,  for  linnets,  and  for 
green-finches;  less  so  for  sparrows.  Chickens,  pigeons,  and  rabbits,  accord- 
ing to  Klein,  are  immune.  Of  eight  mice  inoculated  subcutaneously  with 
one  or  two  drops  of  a  bouillon  culture,  six  died  within  forty-eight  hours 
and  two  recovered.  Out  of  eight  guinea-pigs  inoculated  in  the  same  way 
four  died  in  forty-eight  hours  and  two  recovered.  At  the  autopsy  the 
lungs  and  liver  were  found  to  be  hyperaemic,  the  spleen  not  enlarged.  The 
bacilli  were  present  in  large  numbers  in  blood  from  the  heart  and  in  the 
lungs. 

BACILLUS  GALLINARUM. 

Obtained  by  Klein  (1889)  from  the  blood  of  chickens  which  succumbed 
to  an  epidemic  disease  resembling  "  fowl  cholera."  The  bacillus  is  believed 
by  Klein  not  to  be  identical  with  Pasteur's  bacillus  of  fowl  cholera,  and  is 
said  not  to  be  pathogenic  for  rabbits,  which  would  seem  to  differentiate  it 
from  this  bacillus  (Bacillus  septicaemias  hsemorrhagicae). 

Morphology. — Bacilli  with  rounded  ends,  from  0.8  to  2  u-  long  and 
0.3  to  0.4  it  thick ;  often  in  pairs. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  non-liquefying,  non-motile  bacillus. 
Does  not  form  spores.  Grows  in  the  usual  culture  media  at  the  room  tem- 
perature— better  in  the  incubating  oven.  Upon  gelatin  plates  forms  grayish- 
white,  superficial  colonies,  which  later  present  the  appearance  of  flat,  homo- 
geneous, whitish  discs  with  thin  edges  and  irregular  margins,  and  by 
transmitted  light  have  a  brownish  color.  The  deep  colonies  are  small  and 
spherical,  and  have  a  brownish  color  by  transmitted  light.  In  gelatin  stab 
cultures  a  thin,  gray  layer  with  irregular  margins  and  of  limited  extent 
forms  upon  the  surface,  and  a  scanty  growth  occurs  along  the  line  of  punc- 
ture in  the  form  of  a  grayish-white  line.  Upon  the  surface  of  agar,  at 
37°  C.,  a  thin,  gray  layer  with  irregular  margins  has  developed  at  the  end  of 
twenty-four  hours ;  later  this  extends  over  the  entire  surface  as  a  thin,  gray- 
ish-white layer.  No  growth  occurs  upon  potato  at  37°  C.  In  bouillon,  at  37° 
C.,  development  occurs,  with  clouding  of  the  bouillon,  within  twenty -four 
hours;  later  a  deposit  consisting  of  bacilli  is  seen  at  the  bottom  of  the  tube, 
but  no  film  forms  upon  the  surface. 

Pathogenesis.  — Chickens  inoculated  subcutaneously  with  a  pure  culture 
die  in  from  twenty-four  hours  to  eight  or  nine  days.  Pigeons  and  rabbits 
are  immune. 

BACILLUS   CAPSULATUS. 

Obtained  by  Pfeiffer  (1889)  from  the  blood  of  a  guinea-pig  which  died 
spontaneously. 

Morphology. — Thick  bacilli  with  rounded  ends,  usually  two  or  three 
times  as  long  as  broad;  often  united  in  chains  of  two  or  three  elements;  may 
grow  out  into  homogeneous  filaments.  Stained  preparations  show  the  ba- 
cilli to  be  enveloped  in  an  oval  capsule  which  may  be  considerably  broader 
than  the  bacilli  themselves — two  to  five  times  as  broad ;  where  several  ba- 
cilli are  united  they  are  surrounded  by  a  single  capsular  envelope. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method.  In  pre- 
parations which  are  deeply  stained  with  hot  f  uchsin  or  gentian  violet  solu- 


522 


BACILLI   WHICH   PRODUCE    SEPTICAEMIA 


tion  the  capsule  is  so  deeply  stained  that  the  bacillus  is  hidden;  by  careful 
treatment  with  a  weak  solution  of  acetic  acid  the  capsule  may  be  differen- 
tiated as  a  pale- red  or  violet  envelope  surrounding  the  deeply  stained  bacilli. 

Biological  Characters. — An  aer- 
obic aud  facultative  anaerobic, 
non  liquefying,  non-motile  bacillus. 
Spore  formation  not  observed. 
Grows  in  the  usual  culture  media 
at  the  room  temperature.  The  cul- 
tures in  agar  or  upon  potato  are  very 
viscid  and  draw  out  into  long 
threads  when  touched  with  the  pla- 
tinum needle;  the  blood  of  an  ani- 
mal killed  by  inoculation  with  this 
bacillus  has  the  same  viscid  charac- 
ter. Upon  gelatin  plates  minute 
colonies  are  first  visible  at  the  end 
of  twenty- four  to  thirty-six  hours; 
later  the  deep  colonies  are  white, 
oval  masses  the  size  of  a  pin's  head ; 
the  superficial  colonies  attain  the 
size  of  a  lentil,  and  are  flattened, 
hemispherical  masses  with  a  porce- 
lain-white color.  In  gelatin  stab 
cultures  growth  occurs  to  the  bot- 
tom of  the  line  of  puncture,  and  011 
the  surface  a  shining  white,  circular, 
arched  mass  forms  around  the  point  of  puncture,  resembling  the  growth  of 
Friedlander's  bacillus.  Upon  the  surface  of  agar,  at  37°  C  ,  at  the  end  of 
twenty- four  hours  a  thick,  soft  layer  of  a  pure  white  color  is  formed,  which 
is  very  viscid  and  resembles  the  growth  of  Micrococcus  tetragenus  upon  the 
same  medium.  Upon  potato  an  abundant  and  viscid,  shining,  yellowish- 
white  layer  is  quickly  developed. 

Pathogenesis. — Pathogenic  for  white  mice  and  for  house  mice,  which  die 
at  the  end  of  two  or  three  days  after  being  inoculated  at  the  root  of  the  tail 
with  a  small  quantity  of  a  pure  culture.  Inoculation  from  mouse  to  mouse 
increases  the  virulence  of  the  cultures.  At  the  autopsy  the  superficial  veins 
are  distended  with  blood,  the  inguinal  glands  enlarged,  the  spleen  consid- 
erably enlarged,  the  liver  and  kidneys  hyperaemic,  the  intestine  pale,  the 
heart  distended  with  blood,  which  usually  is  very  viscid  and  is  drawn  out 
into  threads  when  touched  with  the  platinum  needle.  The  bacilli  are  found 
in  the  blood  and  in  all  of  the  organs,  in  the  contents  of  the  peritoneum  and 
pleurae,  and  in  the  exudate  in  the  vicinity  of  the  point  of  inoculation. 
Pathogenic  also  for  guinea  pigs  and  for  pigeons;  guinea-pigs  are  infallibly 
killed  within  thirty-six  hours  by  the  injection  of  a  single  drop  of  a  bouillon 
culture,  twenty-four  hours  old,  into  the  cavity  of  the  abdomen;  the*  blood 
contains  the  bacillus  in  enormous  numbers,  as  does  the  viscid  fluid  found  in 
the  peritoneal  cavity.  Rabbits  do  not  succumb  to  intraperitoneal  or  subcu- 
taneous inoculations,  but  are  killed  by  the  intravenous  injection  of  one 
cubic  centimetre  of  a  recent  bouillon  culture.  Putrefactive  changes  occur 
very  quickly  in  animals  killed  by  inoculation  with  this  bacillus. 


FIG  141.— Bacillus  capsulatus,  from  peritoneal 
exudate  of  an  inoculated  guinea-pig.  X  1,000. 
From  a  photomicrograph.  (Ffeiffer.) 


BACILLUS   HYDROPHILUS   FUSCUS. 

Obtained  by  Sanarelli  (1891)  from  the  lymph  of  frogs  suffering  from  a 
fatal  infectious  disease. 

Morphology. — Bacilli  with  rounded  ends,  usually  from  1  to  3  /*  in  length; 
often  short  oval ;  may  grow  out  into  filaments  of  12  to  20  /*  in  length. 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacillus.  Grows 
in  the  usual  culture  media  at  the  room  temperature.  In  gelatin  stab  cul- 


IN   SUSCEPTIBLE   ANIMALS. 


523 


FIG.  143.— Bacillus  hydroph'lus  fus- 
cus,  in  blood  of  triton.    (Sanarelli.) 


tures,  at  18°  to  20'  C.,  liquefaction  has  already  commenced  along-  the  line  of 
puncture  at  the  end  of  twelve  hours,  and  at  the  end  of  thirty-six  to  forty- 
eight  hours  half  of  the  gelatin  is  liquefied  iu  funnel  shape ;  on  the  third  or 
fourth  day  the  gelatin  is  completely  lique- 
fied, and  a  thick,  white,  flocculent  deposit 
is  seen  at  bottom  of  the  tube.  In  glycerin- 
agar,  at  37°  C.,  a  slight,  bluish,  diffuse 
fluorescence  is  seen  upon  the  surface  at  the 
end  of  twelve  hours,  and  soon  after  a  luxu- 
riant growth,  which  soon  covers  the  entire 
surface,  is  developed ;  at  the  end  of  twenty- 
four  to  thirty-six  hours  large  gas  bubbles 
begin  to  form  in  the  agar;  gradually  the 
fluorescence  disappears,  the  surface  growth 
becomes  thicker  and  has  a  dirty -gray  color 
which  changes  later  to  brownish.  Blood 
serum  is  a  favorable  medium  and  is  rapidly 
liquefied  by  this  bacillus.  Upon  potato  the 
growth  is  most  characteristic.  At  the  end 
of  twelve  hours  a  thin,  straw-yellow  layer 
is  developed  along  the  impfstrich;  this 
gradually  becomes  yellow,  and  at  the  end 
of  four  to  five  days  has  a  brown  color,  resembling  that  of  the  glanders  bacil- 
lus upon  potato. 

Pathogenesis. — Pathogenic  for  frogs,  toads,  lizards,  and  oth  "cold- 
blooded" animals;  also  for  guinea-pigs,  rabbits,  dogs, 
cats,  mice,  chickens,  and  pigeons.  When  a  few  drops  of 
a  bouillon  culture  are  injected  into  the  muscles  of  the 
thigh,  swelling  and  redness  at  the  point  of  inoculation 
are  quickly  developed,  and  death  usually  occurs  in  eight 
to  ten  hours.  The  bacilli  are  found  in  great  numbers  in 
the  blood  and  in  all  of  the  organs.  Guinea  pigs  die  from 
general  infection  within  twelve  hours  after  receiving  a 
subcutaneous  injection  of  a  small  amount  of  a  pure  cul- 
ture; the  spleen  is  enlarged  and  the  liver  and  spleen  hy- 
peraemic;  an  extensive  inflammatory  oedema  in  the  vicin- 
ity of  the  inoculation  wound  is  frequently  observed;  the 
bacilli  are  very  numerous  in  the  blood  and  in  all  the  or- 
gans. Rabbits  die  in  five  to  six  hours  from  an  intravenous 
injection.  Adult  dogs  are  immune,  but  new-born  dogs 
(tnree  to  four  days  old)  die  infallibly,  after  receiving  a 
subcutaneous  injection  of  a  small  quantity  of  a  pure  cul- 
ture, in  twelve  to  thirty-six  hours.  Young,  cats  also  suc- 
cumb to  similar  inoculations.  Chickens  and  pigeons  die 
within  five  to  seven  hours  after  receiving  an  intravenous 
injection,  but  resist  subcutaneous  injections. 


FIG.  143.— Bacillus 
hydrophilus  fuscus; 
culture  in  nutrieut 
gelatin,  end  of  six- 
teen hours.  (Sana- 
relli.) 

Pathogenesis. 
pigs  or  for  white 


BACILLUS   TENTHS   SPUTIGENUS. 

Obtained  by  Pansini  (1890)  from  sputum. 

Morphology.  Short  bacilli,  usually  in  pairs  and  sur- 
rounded by  a  capsule. 

Stains  by  Gram's  method. 

Biological  Characters. — An  aerobic,  non-liquefying, 
non  motile  bacillus.  Grows  in  nutrient  gelatin  at  the 
room  temperature.  Develops  abundantly  on  potato. 
Coagulates  milk  and  produces  an  acid  reaction  in  this 
medium. 

—  Pathogenic  for  rabbits  and  white  rats ;  not  for  guinea- 
mice  (in  small  doses). 


524  BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

BACILLUS   OF  LASER. 

Obtained  by  Laser  (1892)  from  mice  which  succumbed  to  an  epidemic  dis 
ease  in  Frankel's  laboratory  at  Konigsberg. 

In  its  characters  this  bacillus  closely  resembles  the  bacillus  of  swine 
plague,  and  is  perhaps  identical  with  it. 

Morphology. — A  small  bacillus,  with  rounded  ends,  about  twice  as  long 
as  broad.  Has  fiagella  both  at  the  extremities  and  sides. 

Stains  by  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  actively  motile  bacillus.  Spore  formation  not  observed.  Grows 
either  in  the  incubating  oven  or  at  the  room  temperature.  Thermal  death - 
point  65°  to  70°  C. — ten  minutes'  exposure.  Upon  gelatin  plates,  at  the  end 
of  two  days,  the  deep  colonies  are  spherical,  finely  granular,  and  brownish 
in  color;  the  superficial  are  transparent,  finely  granular,  and  leaf -like. 
In  gelatin  stab  cultures  growth  occurs  along  the  entire  line  of  puncture  as 
well  as  upon  the  surface.  At  the  end  of  three  days  a  considerable  evolution 
of  gas  is  usually  observed.  In  agar  an  abundant  development  is  seen  at  the 
end  of  twenty- four  hours  in  the  incubating  oven;  upon  the  surface  a  gray- 
ish-white, shining  layer  with  dentate  margins  is  formed  along  the  track  of 
the  needle.  In  bouillon,  at  37°  C.,  development  is  abundant  and  rapid;  a 
thin  film  is  formed  on  the  surface  at  the  end  of  the  second  day.  Upon  potato 
a  brownish  layer  is  formed  at  the  end  of  twenty-four  hours.  In  milk  an 
acid  reaction  is  produced. 

Pathogenesis. — Pathogenic  for  field  mice,  guinea-pigs,  rabbits,  and 
pigeons.  The  bacillus  is  found  in  the  blood  and  various  organs  of  infected 
mice.  The  spleen  is  found  to  be  greatly  enlarged. 

BACILLUS  TYPHI  MURIUM  (Loffler). 

Obtained  by  Loffler  (1889)  from  mice  which  died  in  his  laboratory  from 
an  epidemic  disease  due  to  this  bacillus. 

Morphology. — Short  bacilli,  resembling  the  bacillus  of  diphtheria  in 
pigeons,  and  varying  considerably  in  dimensions— like  the  bacillus  of 
typhoid  fever;  grows  out  in  to  flexible  filaments. 

Stains  with  the  aniline  colors— best  with  Loffler's  solution  of  methylene 
blue. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Spore  formation  not  determined.  Has  flagella 
around  the  periphery  of  the  cells,  like  those  of  the  typhoid  bacillus,  and  ex- 
hibits similar  active  movements.  In  gelatin  stab  cultures,  at  the  room 
temperature,  growth  occurs  upon  the  surface,  at  the  end  of  forty -eight  hours, 
in  the  form  of  a  flat,  grayish- white,  round,  semi-transparent  mass  the  size  of 
a  pin's  head ;  later  the  surface  colony  increases  in  extent  and  has  more  or 
less  irregular  margins.  In  gelatin  plate  cultures  the  deep  colonies  are  at 
first  round,  slightly  granular,  transparent,  and  grayish;  later  they  are  of  a 
yellowish- brown  color  and  decidedly  granular.  The  superficial  colonies  are 
very  granular  and  marked  by  delicate  lines — similar  to  colonies  of  the 
typhoid  bacillus.  Upon  agar  a  grayish- white  layer  is  developed  which  is 
not  at  all  characteristic.  Upon  potato  a  rather  thin,  whitish  layer  is  formed, 
and  around  this  the  potato  acquires  a  dirty  bluish-gray  color.  In  milk  an 
abundant  development  occurs,  and  a  decidedly  acid  reaction  is  produced 
without  causing  any  perceptible  change  in  the  appearance  of  the  fluid. 

Pathogenesis.—  Pathogenic  for  white  mice,  which  die  in  from  one  to  two 
weeks  after  infection  ;  also  to  field  mice,  which  succumb  to  subcutaneous  in- 
jections of  a  pure  culture,  and  also,  in  from  eight  to  twelve  days,  when  fed 
upon  potato  cultures  or  bread  moistened  with  a  small  quantity  of  a  bouillon 
culture.  Loffler  believes  that  this  bacillus  may  be  used  for  the  destruction 
of  field  mice  in  grain  fields,  inasmuch  as  they  invariably  die  after  ingesting 
food  which  has  been  contaminated  with  it,  and  also  from  eating  the  bodies 


IN    SUSCEPTIBLE   ANIMALS.  525 

of  other  mice  which  have  died  as  a  result  of  infection.  House  mice  are  also 
susceptible.  Rabbits,  guinea  pig's,  pig-eons,  and  chickens  were  found  by 
Loffler  not  to  be  susceptible  to  infection  by  feeding. 

BACILLUS  OF  CAZAL  AND  VAILLARD. 

Obtained  by  Cazal  and  Vaillard  (1891)  from  cheesy  nodules  upon  the 
peritoneum  and  in  the  pancreas  of  an  individual  who  died  in  the  hospital 
at  Val  de  Grace. 

Morphology. — Bacilli  with  rounded  ends,  but  little  longer  than  they  are 
broad ;  solitary,  in  pairs,  or  in  chains  of  ten  to  fifteen  or  more  elements. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method ;  the 
extremities  of  the  rods  are  more  deeply  stained  than  the  central  portion— 
44  polar  staining." 

Biological  Characters. — An  aerobic  find  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Does  not  form  spores.  Grows  in  the  usual  culture  media 
at  the  room  temperature — more  rapidly  in  the  incubating  oven  at  37°  C.  In 
gelatin  stab  cultures,  at  the  end  of  twenty-four  hours,  a  series  of  puncti- 
f  orm,  white  colonies  is  developed  along  the  line  of  puncture ;  upon  the  sur- 
face development  is  more  abundant,  and  at  the  end  of  forty-eight  hours 
liquefaction  commences  ;  this  progresses  slowly  from  above  downward, 
and  a  white,  flocculent  deposit  accumulates  at  the  bottom  of  the  liquefied 

Statin.  ^Upon  the  surface  of  agar,  at  the  end  of  twenty-four  hours  at  37° 
.,  a  moist,  transparent,  opalescent  layer  is  developed,  which  rapidly  ex- 
tends over  the  entire  surface  ;  later  this  layer  becomes  somewhat  thicker, 
whitish,  and  cream- like  in  consistence,  without  losing  its  transparency. 
Upon  potato  a  thick,  prominent,  moist,  and  slightly  viscid  layer  is  devel- 
oped, which  at  first  has  a  pale-yellow  and  later  a  yellowish-brown  color. 
In  bouillon  development  is  abundant,  producing  a  milky  opacity  of  the 
liquid;  a  thick,  flocculent  deposit  accumulates  at  the  bottom  of  the  tube  ; 
the  reaction  of  the  culture  liquid  becomes  very  alkaline.  All  of  the  cultures 
give  off  a  peculiar  odor,  slightly  ammoniacal  and  resembling1  that  of  putrid 
urine.  The  cultures  retain  their  vitality  for  several  months — in  a  closed 
tube  for  more  than  a  year.  The  thermal  death-point  is  60°  C.  with  fifteen 
minutes'  exposure. 

Pathogenesis. — Pathogenic  for  rabbits  and  mice,  but  not  for  guinea-pigs. 
In  mice  death  occurs  from  general  infection,  at  the  end  of  forty-eight  to 
sixty  hours,  from  the  subcutaneous  injection  of  one  eighth  cubic  centimetre 
of  a  recent  bouillon  culture.  In  rabbits  injection  of  one  cubic  centimetre 
into  the  circulation  causes  the  death  of  the  animal  in  thirty-six  to  fifty 
hours.  The  symptoms  induced  are  a  foetid  diarrhoea  and  paralysis  of  the 
extremities.  When  smaller  doses  are  injected  (0.5  cubic  centimetre)  a 
chronic  malady  is  developed,  characterized  at  the  outset  by  diarrhoea  and 
emaciation,  then  by  the  development  of  tumors  which  resemble  those  found 
in  the  man  from  whom  the  cultures  were  first  obtained.  These  tumors  are 
for  the  most  part  located  in  the  subcutaneous  connective  tissue ;  after  a  time 
they  attain  the  size  of  a  chestnut  and  ulcerate,  allowing  the  escape  of  a 
semi-fluid,  purulent  material.  The  animals  usually  recover.  Similar  tumors 
are  developed  as  a  result  of  subcutaneous  injections  of  one  to  three  cubic 
centimetres  of  a  recent  bouillon  culture. 

BACILLUS  OF  BABES  AND  OPRESCU. 

Obtained  by  Babes  and  Oprescu  (1891)  from  a  case  of  septicaemia  hsemor- 
rhagica  presenting  some  resemblance  to  exanthematic  typhus. 

Morphology.— In  agar  cultures  the  bacilli  are  from  0.4  to  0.5  jj.  thick,  and 
are  frequently  united  in  pairs ;  associated  with  these  rod-shaped  bacteria  are 
forms  which  are  of  a  short  oval.  In  gelatin  cultures  oval  forms  are  more 
numerous  ;  they  have  a  diameter  of  0.3  to  0.4  JLI,  and  often  appear  to  be 
surrounded  by  a  capsule.  In  fresh  cultures  the  bacilli  are  often  in  form  of 


BACILLI   WHICH   PRODUCE   SEPTICAEMIA 

a  figure  8,  and  are  only  stained  at  the  point  of  contact  of  the  two  segments. 
In  potato  cultures  they  are  sometimes  elongated  and  swollen  at  one  ex- 
tremity. 

Stains  with  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, actively  motile  bacillus.  Spore  formation  not  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature — more  rapidly  at  37°  C.  In 
gelatin  stab  cultures  yellowish- white  colonies  are  developed  along  the  line 
of  puncture;  at  the  bottom  these  may  have  a  diameter  of  one  to  two  millime- 
tres, and  they  have  a  brown  color.  Upon  the  surface  an  irregular,  lobulated, 
whitish,  translucent,  paraffin  like  layer  is  developed.  At  the  end  of  eight 
days  the  surface  growth  consists  of  large,  confluent,  transparent  plaques, 
with  irregular  outlines  and  crenated,  elevated  margins  ;  along  the  line  of 
puncture  large,  separate,  lenticular  or  spherical  colonies  are  seen  ;  these 
have  a  brownish-white  color.  At  the  end  of  two  months  the  surface  growtli 
is  concentric  and  still  more  transparent,  while  the  colonies  near  the  surface 
have  become  almost  brown.  Upon  the  surface  of  agar,  at  37°  C.,  a  narrow 
band  is  developed  along  the  line  of  inoculation ;  above,  this  is  composed  of 
transparent,  shining,  flat,  round  colonies  having  a  diameter  of  one  milli- 
metre or  more ;  below,  the  colonies  are  confluent  and  form  a  transparent, 
whitish  layer.  In  glycerin-agar  development  is  still  more  abundant,  and 
may  already  be  perceived  at  the  end  of  twelve  hours.  Crystals  are  seen 
below  the  surface  in  agar  cultures  and  about  the  super ficial  colonies  in  gela- 
tin. Upon  potato  a  uniform,  thin,  grayish,  very  transparent  layer  is  de- 
veloped, which  sometimes  has  a  brownish-gray  tint.  At  the  end  of  a  few 
days  the  potato  acquires  a  brownish  color.  In  bouillon  cloudiness  of  the 
medium  is  apparent  at  the  end  of  ten  hours  ;  twenty-four  hours  later  a 
whitish  precipitate  is  seen  at  the  bottom  of  the  tube,  which  is  more  abun- 
dant when  the  culture  medium  contains  glucose;  later  a  thin  pellicle  is 
seen  upon  the  surface  and  the  bouillon  acquires  a  yellowish  color. 

Pathogenesis. — Recent  cultures  are  pathogenic  for  rabbits,  guinea-pigs, 
pigeons,  and  mice,  which  die  from  general  infection  in  from  two  to  four 
days.  Old  cultures  are  less  virulent. 

BACILLUS   OF  LUCET. 

Obtained  by  Lucet  (1891)  from  chickens  and  turkeys  suffering  from  an 
infectious  form  of  septicaemia  characterized  by  dysenteric  discharges — "  Dy- 
seiiterie  epizootique  des  poules  et  des  dindes." 

Resembles  Bacillus  gallinarum  of  Klein,  and  is  perhaps  identical  with 
this  microorganism. 

Morphology. — Short  bacilli,  from  1.2  to  1.8  fit  long,  usually  in  pairs. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non-lique- 
fying, non-motile  bacillus.  Spore  formation  not  observed.  Grows  slowly  in 
the  usual  culture  media  at  the  room  temperature — more  rapidly  at  37°  0. 

In  gelatin  plates  small,  shining,  moist,  white,  circular  colonies  are  devel- 
oped, which  look  like  little  drops  of  wax ;  later  these  increase  in  size,  and 
especially  in  thickness,  forming  hemispherical  masses.  In  gelatin  stab  cul- 
tures grayish,  punctiform  colonies  are  developed  along  the  line  of  puncture, 
and  upon  the  surface  a  circular,  prominent,  whitish  plaque.  Streak  cultures 
upon  the  surface  of  gelatin  are  in  the  form  of  a  dirty- white  or  grayish- white, 
moist  streak,  with  regular  margins,  limited  to  the  line  of  inoculation,  but 
increasing  in  thickness  until  it  breaks  loose  and  slips  down  the  oblique  sur- 
face of  the  culture  medium.  The  deposit  which  collects  in  this  way  acquires, 
as  it  becomes  old,  in  the  deepest  portion  a  reddish  color.  Upon  agar  it  forms 
a  thick,  yellowish-white,  mucus-like  layer  with  straight  or  slightly  dentate 
margins.  In  bouillon  it  produces  a  decided  clouding  of  the  liquid,  and  an 
abundant  grayish,  pulverulent  sediment  accumulates  at  the  bottom  of  th^ 
tube ;  the  bouillon  after  a  time  becomes  transparent  above  this  sediment  and 


PLATE   VII. 

BACILLUS     OF    GLANDERS. 

FIG.  1. — Bacillus  mallei  from  the  liver  of  a  field  mouse,  cover-glass 
preparation.  (Loffler.) 

FIG.  2. — Bacillus  mallei  from  a  recent  culture  upon  blood  serum. 
(Loffler.) 

FIG.  3. — Bacillus  mallei  in  section  of  spleen  of  a  field  mouse  dead 
from  glanders.  (Loffler.) 

FIG.  4. — Culture  of  glanders  bacillus  upon  cooked  potato.     (Loffler.) 


STERNBERG'S  BACTERIOLOGY. 


Fig.l. 


Fig.  2. 


BACILLUS  OF  GIANDERS  (LOEFFLER) 


IN   SUSCEPTIBLE   ANIMALS.  527 

is  viscid,  drawing  out  into  threads.  In  the  absence  of  oxygen  the  characters 
of  growth  are  the  same  as  in  its  presence.  The  cultures  acquire  an  alkaline 
reaction ;  they  are  sterilized  by  exposure  for  ten  minutes  to  a  temperature  of 
60°  C.  Does  not  grow  upon  potato. 

Pathogenesis. — Pathogenic  for  chickens  and  turkeys.  Not  pathogenic  for 
pigeons,  guinea-pigs,  or  rabbits  when  injected  subcutaiieously  or  into  the 
peritoneal  cavity,  but  kills  rabbits  when  injected  into  a  vein.  In  the  in- 
fected fowls  the  bacilli  are  found  in  small  numbers  in  the  blood,  more  nu- 
merous in  the  kidneys  and  liver,  still- more  numerous  in  the  spleen,  and  in 
enormous  numbers  in  the  intestinal  mucus,  where  in  acute  cases  it  is  found 
almost  in  a  pure  culture.  Fowls  do  not  contract  the  disease  as  a  result  of 
the  ingestioii  of  grains  soiled  with  cultures  of  the  bacillus,  but  become  in- 
fected when  fed  with  animal  food  to  which  a  pure  culture  has  been  added. 


XIV. 

PATHOGENIC  AEROBIC  BACILLI    NOT  DESCRIBED  IN 

PREVIOUS  SECTIONS. 

A  CONSIDERABLE  number  of  saprophytic  bacilli  are  pathogenic  for 
small  animals  when  injected  into  the  circulation,  or  subcutaneously, 
or  into  a  serous  cavity  in  considerable  quantity — one  to  five  cubic 
centimetres  or  more — but  fail  to  produce  any  appreciable  effect 
when  introduced  into  the  bodies  of  these  animals  in  minute  doses, 
and  do  not  multiply  in  the  blood  to  any  considerable  extent,  al- 
though in  fatal  cases  they  may  usually  be  recovered  in  cultures  from 
the  blood  and  tissues.  These  bacilli  are  pathogenic  by  reason  of  the 
toxic  ptomaines  produced  by  them,  or  because  of  local  inflammatory 
processes  which  they  induce,  or  for  both  of  these  reasons  combined. 
Some  of  them  may  also,  under  certain  circumstances,  multiply  in 
the  blood  and  thus  give  rise  to  septicaemia  as  well  as  to  toxaemia ; 
this  is  the  case,  for  example,  with  the  "  colon  bacillus  "  of  Escher- 
ich.  When  injected  in  considerable  quantity  into  the  circulation 
of  a  guinea-pig  it  causes  the  death  of  the  animal  within  twenty-four 
hours,  and  the  bacillus  is  found  in  the  blood  in  great  numbers  ;  but 
minute  amounts  injected  into  a  vein,  or  larger  amounts  injected 
subcutaneousiy,  do  not  usually  produce  general  infection.  It  is, 
therefore,  not  included  among  the  "bacilli  which  produce  septi- 
caemia in  susceptible  animals."  There  is  reason  to  believe,  however, 
that  under  certain  circumstances  this  bacillus  may  have  sufficient 
pathogenic  potency  to  produce  a  genuine  septicaemia  in  guinea-pigs. 
Thus  the  original  cultures  of  Brieger's  bacillus,  which  appears  to  be 
a  variety  of  the  colon  bacillus,  are  reported  to  have  produced  fatal 
septicaemia  in  guinea-pigs  when  injected  subcutaneousiy  in  small 
amounts.  A  strict  division  into  pathogenic  bacilli  which  produce 
general  blood  infection — septicaemia — and  those  which  produce  a 
fatal  result  owing  to  the  production  of  toxic  chemical  substances  is 
not  possible;  for  many  pathogenic  bacteria  produce  general  infection 
when  injected  in  comparatively  large  doses,  and  at  the  same  time 
give  rise  to  symptoms  of  toxaemia  ;  or  general  infection  may  occur 
in  animals  of  one  species,  and  fatal  toxaemia  without  septicaemia  in 


PATHOGENIC   AEROBIC   BACILLI   NOT  BEFORE   DESCRIBED.       529 

those  of  another  species.  Many  of  the  bacilli  described  in  the  pre- 
sent section  are  common  saprophytes,  which  have  been  shown  by 
laboratory  experiments  to  be  pathogenic  for  certain  animals  when 
introduced  into  their  bodies  in  a  certain  amount,  which  differs  greatly 
for  different  bacteria  and  for  different  species  of  animals.  The  ex- 
periments of  Cheyne  and  others  show  how  largely  the  pathogenic 
power  of  saprophytic  bacteria  depends  upon  the  quantity  of  a  cul- 
ture which  is  injected,  as  well  as  upon  the  age  of  the  culture  and 
the  seat  of  the  inoculation — in  the  blood,  the  abdominal  cavity,  the 
subcutaneous  tissues,  or  the  muscles.  And  the  bacteriologist  named 
has  also  shown  that  pathogenic  power  depends,  in  some  instances  at 
least,  upon  the  combined  action  of  the  toxic  substances  introduced 
in  the  first  instance  and  of  the  living  bacteria.  Thus  Cheyne  found 
that  one-tenth  of  a  cubic  centimetre  of  a  bouillon  culture  of  Proteus 
vulgaris  injected  into  the  dorsal  muscles  of  a  rabbit  infallibly  caused 
its  death  within  forty-eight  hours,  but  when  the  dose  was  reduced 
to  one-fortieth  cubic  centimetre  the  animal  recovered.  But  if  to 
this  amount  (one-fortieth  cubic  centimetre)  he  added  one  cubic  cen- 
timetre of  a  sterilized  (by  heat)  culture  of  the  same  bacillus  instead 
of  diluting  with  distilled  water,  and  injected  the  mixture  into  the 
dorsal  muscles  of  a  rabbit,  death  occurred  in  every  experiment 
within  forty-eight  hours.  The  sterilized  culture  injected  by  itself 
produced  no  effect  in  this  dose  (one  cubic  centimetre),  and  Cheyne 
believes  that  the  fatal  result  in  these  experiments  was  due  to  the 
fact  that  the  toxic  products  present  in  the  sterilized  culture  over- 
came the  natural  resisting  powers  of  the  tissues  and  enabled  the 
bacillus  to  multiply  over  a  larger  area  than  would  otherwise  have 
been  the  case.  As  a  result  of  this,  toxic  substances  were  produced  in 
the  body  of  the  animal  in  sufficient  quantity  to  cause  general  toxae- 
mia and  death  ;  whereas  the  bacilli  alone,  in  the  dose  mentioned, 
were  not  able  to  invade  the  tissues  in  the  vicinity  of  the  point  of 
inoculation,  and  gave  rise  to  a  local  abscess  only.  The  same  ex- 
planation is  probably  true  for  very  many  of  the  saprophytic  bacteria 
which  have  been  shown  to  possess  pathogenic  power  ;  and  it  is  prob- 
able that  many  of  those  which  are  now  classed  by  bacteriologists  as 
non-pathogenic  would  prove  to  be  pathogenic  in  the  same  way  if 
thoroughly  tested  upon  various  species  of  animals,  although  it  might 
be  necessary  to  use  unusually  large  doses  to  accomplish  the  same 
result. 

BACILLUS   COLI   COMMUNIS. 

Synonyms. — Bacterium  coli  commune  (Escherich);  Colon  bacillus 
of  Escherich ;  Emmerich's  bacillus  (Bacillus  Neapolitanus).     Prob< 
ably  identical  with  Bacillus  cavicida  (Brieger's  bacillus). 
34 


530  PATHOGENIC   AEROBIC   BACILLI 

Obtained  by  Emmerich  (1885)  from  the  blood,  various  organs,  and 
the  alvine  discharges  of  cholera  patients  at  Naples  ;  by  Weisser 
(1886)  from  normal  and  abnormal  human  faeces,  from  the  air,  and 
from  putrefying  infusions  ;  by  Escherich  (1886)  from  the  faeces  of 
healthy  children  ;  since  shown  to  be  commonly  present  in  the  alvine 
discharges  of  healthy  men,  and  probably  of  many  of  the  lower  ani- 
mals. Found  by  the  writer  in  the  blood  and  various  organs  of  yellow- 
fever  cadavers,  in  Havana  (1888  and  1889). 

Numerous  varieties  have  been  cultivated  by  different  bacteriolo- 
gists, which  vary  in  pathogenic  power  and  to  some  extent  in  their 
growth  in  various  culture  media  ;  but  the  differences  described  are 
not  sufficiently  characteristic  or  constant  to  justify  us  in  considering 
them  as  distinct  species. 

Morphology.  —  Differs  considerably  in  its  morphology  as  obtained 

from  different  sources  and  in  various  culture  media.     The  typical 

form  is  that  of  short  rods  with  rounded  ends,  from  two  to  three  >w  in 

length  and  0.4  to  0.6  /^  broad  ;  but  under  certain  cir- 

«  «^  ^          cumstances  the  length  does  not  exceed  the  breadth— 
ff  WJ/  J  ^     about  0.5  fj.  —  and  it  might  be  mistaken  for  a  micrococ- 
cus  ;  again  the  prevailing  form  in  a  culture  is  a  short 


oval  ;  filaments  of  five  /*  or  more  in  length  are  often 
FIG.   144.—  Ba-  observed  in  cultures,  associated  with  short  rods  or  oval 
emus    coii  com-      u        The  bacilli  are  frequently  united  in  pairs.     The 

munis.      X    1  000.  «_ 

(Escherich.)  presence  of  spores  has  not  been  demonstrated.  In  un- 
favorable culture  media  the  bacilli,  in  stained  prepara- 
tions, may  present  unstained  places,  which  are  supposed  by  Escherich 
to  be  due  to  degenerative  changes  in  the  protoplasm.  Under  certain 
circumstances  some  of  the  rods  in  a  pure  culture  have  been  observed 
by  Escherich  to  present  spherical,  unstained  portions  at  one  or  both 
extremities,  which  closely  resemble  spores,but  which  he  was  not  able 
to  stain  by  the  methods  usually  employed  for  staining  spores,  and 
which  he  is  inclined  to  regard  as  "  involution  forms." 

This  bacillus  stains  readily  with  the  aniline  colors  usually  em- 
ployed by  bacteriologists,  but  quickly  parts  with  its  color  when 
treated  with  iodine  solution  —  Gram's  method  —  or  with  diluted  al- 
cohol. 

Biological  Characters.  —  An  aerobic  and  facultative  anaerobic, 
non-liquefying  bacillus.  Sometimes  exhibits  independent  move- 
ments, which  are  not  very  active.  One  rod  of  a  pair,  in  a  hanging- 
drop  culture,  may  advance  slowly  with  a  to-and-fro  movement, 
while  the  other  follows  as  if  attached  to  it  by  an  invisible  band 
(Escherich).  The  writer's  personal  observations  lead  him  to  believe 
that,  as  a  rule,  this  bacillus  does  not  exhibit  independent  movements. 
Does  not  form  spores.  Grows  in  various  culture  media  at  the  room 


NOT    DESCRIBED    IN    PREVIOUS   SECTIONS.  531 

temperature — more  rapidly  in  the  incubating  oven.     Grows  in  a  de- 
cidedly acid  medium. 

In  gelatin  plates  colonies  are  developed  in  from  twenty-four  to 
forty-eight  hours,  which  vary  considerably  in  their  appearance  ac- 
cording to  their  age,  and  in  different  cultures  in  the  same  medium. 
The  deep  colonies  are  usually  spherical  and  at  first  are  transparent, 
homogeneous,  and  of  a  pale-straw   or  amber  color  by  transmitted 
light ;  later  they  frequently  have  a  dark-brown,  opaque  central  por- 
tion surrounded  by  a  more  transparent  peripheral  zone  ;  or  they  may 
be  coarsely  granular  and  opaque ;  sometimes  they  have  a  long-oval 
or  "whetstone"  form.     The  superficial  colonies  differ  still  more  in 
appearance  ;  very  young  colonies  by  transmitted  light  often  resemble 
little  drops  of  water  or  fragments  of  broken  glass  ;  when  they  have 
sufficient  space  for  their  development  they  quickly  increase  in  size, 
and  may  attain  a  diameter  of  three  to  four  centimetres  ;  the  central 
portion  is  thickest,  and  is  often  marked  by  a  spherical  nucleus  of  a 
dark-brown  color  when  the  colony  has  started  below  the  surface  of 
the   gelatin  ;  the  margins  are  thin  and  transparent,  the  thickness 
gradually  increasing  towards  the  centre,  as  does  also  the  color,  which 
by  transmitted  light  varies  from  light  straw  color  or  amber  to  a  dark 
brown.     The  outlines  of  superficial  colonies  are  more  or  less  irregular, 
and  the  surface  may  be  marked  by  ridges,  fissures,  or  concentric 
rings,  or  may  be  granular.     The  writer  has  observed  colonies  re- 
sembling a  rosette,  or  a  daisy  with  expanded  petals.     Escherich 
speaks  of  colonies  which  present  star-shaped  figures  surrounded  by 
concentric  rings. 

In  gelatin  stab  cultures  the  growth  upon  the  surface  is  rather 
dry,  and  may  be  quite  thin,  extending  over  the  entire  surface  of  the 
gelatin,  or  it  may  be  thicker  with  irregular,  leaf -like  outlines  and 
with  superficial  incrustations  or  concentric  annular  markings.  An 
abundant  development  occurs  all  along  the  line  of  puncture,  which 
in  the  deeper  portion  of  the  gelatin  is  made  up  of  more  or  less  closely 
crowded  colonies  ;  these  are  white  by  reflected  light,  and  of  an  am- 
ber or  light-brown  color  by  transmitted  light ;  later  they  may  become 
granular  and  opaque.  Frequently  a  diffused  cloudy  appearance  i& 
observed  near  the  surface  of  the  gelatin,  and  under  certain  circum- 
stances branching,  moss-like  tufts  develop  at  intervals  along  the  line 
of  growth.  One  or  more  gas  bubbles  may  often  be  seen  in  recent 
stick  cultures  in  gelatin. 

Upon  nutrient  agar  and  blood  serum,  in  the  incubating  oven,  an 
abundant,  soft,  white  layer  is  quickly  developed.  Upon  potato  an 
abundant,  soft,  shining  layer  of  a  brownish-yellow  color  is  developed. 
The  growth  upon  potato  differs  considerably,  according  to  the  age  of 
the  potato.  According  to  Escherich,  upon  old  potatoes  there  may 


532 


PATHOGENIC   AEROBIC   BACILLI 


be  no  growth,  or  it  may  be  scanty  and  of  a  white  color.  In  milk,  at 
37°  C.,  an  acid  reaction  and  coagulation  of  the  casein  are  produced  at 
the  end  of  eight  or  ten  days.  In  the  absence  of  oxygen  this  bacillus 
is  able  to  grow  in  solutions  containing  grape  sugar  (Escherich).  In 
bouillon  it  grows  rapidly,  producing  a  milky  opacity  of  the  culture 
liquid.  The  thermal  death-point  of  Emmerich's  bacillus,  and  of  the 
colon  bacillus  from  faeces,  was  found  by  Weisser  to  be  60°  C.,  the 
time  of  exposure  being  ten  minutes.  The  writer  has  obtained  corre- 
sponding results.  Weisser  found  that  when  the  bacilli  from  a  bouil- 
lon culture  were  dried  upon  thin  glass  covers  they  failed  to  grow 


Fio.  145.  FIG.  146. 

Fia.  145. — Bacillus  coli  communis  in  nutrient  gelatin  containing  twenty  per  cent  of  gelatin,  end 
of  two  weeks,  showing  moss-like  tufts  along  the  line  of  growth.  (Sternberg.) 

FIG.  146.— A  portion  of  the  growth  shown  in  Fig  147,  at  a,  magnified  about  six  diameters. 
From  a  photograph.  (Sternberg.) 

after  twenty-four  hours.  These  results  give  confirmation  to  the 
view  that  the  bacillus  under  consideration  does  not  form  spores. 
This  view  receives  further  support  from  the  experiments  of  Wal- 
liczek  (1894),  who  found  that  when  dried  upon  pieces  of  sterile  filter 
paper  the  bacillus  failed  to  grow  at  the  end  of  eighteen  hours. 

Pathogenesis. — Comparatively  small  amounts  of  a  pure  culture 
of  the  colon  bacillus  injected  into  the  circulation  of  a  guinea-pig 
usually  cause  the  death  of  the  animal  in  from  one  to  three  days,  and 
the  bacillus  is  found  in  considerable  numbers  in  its  blood.  But  when 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  533 

injected  subcutaneously  or  into  the  peritoneal  cavity  of  rabbits  or 
guinea-pigs,  a  fatal  termination  depends  largely  on  the  quantity  in- 
jected ;  and  although  the  bacillus  may  be  obtained  in  cultures  from 
the  blood  and  the  parenchyma  of  the  various  organs,  it  is  not  present 
in  large  numbers,  and  death  appears  to  be  due  to  toxaemia  rather  than 
to  septicaemia.  Mice  are  not  susceptible  to  infection  by  subcutaneous 
injections.  Small  quantities  injected  beneath  the  skin  of  guinea-pigs 
usually  produce  a  local  abscess  only  ;  larger  amounts — two  to  five 
cubic  centimetres — frequently  produce  a  fatal  result,  with  symptoms 
and  pathological  appearances  corresponding  with  those  resulting 
from  intravenous  injection.  These  are  fever,  developed  soon  after 
the  injection,  diarrhoea,  and  symptoms  of  collapse  appearing  shortly 
before  death.  At  the  autopsy  the  liver  and  spleen  appear  normal,  or 
nearly  so ;  the  kidneys  are  congested  and  may  present  scattered 
punctiform  ecchymoses  (Weisser).  According  to  Escherich,  the 
spleen  is  often  somewhat  enlarged.  The  small  intestine  is  hyper- 
aemic,  especially  in  its  upper  portion,  and  the  peritoneal  layer  pre- 
sents a  rosy  color ;  the  mucous  membrane  gives  evidence  of  more 
or  less  intense  catarrhal  inflammation,  and  contains  mucus,  often 
slightly  mixed  with  blood.  In  rabbits  death  occurs  at  a  somewhat 
later  date,  and  diarrhoea  is  a  common  symptom.  In  dogs  the  subcu- 
taneous injection  of  a  considerable  quantity  of  a  pure  culture  may 
give  rise  to  an  extensive  local  abscess. 

In  human  pathology  the  colon  bacillus  plays  an  important  role. 
It  is  concerned  in  the  etiology  of  a  considerable  proportion  of  the 
cases  of  cystitis  and  of  pyelonephritis,  and  peritonitis  resulting  from 
perforation.  It  appears  to  be  the  cause  of  certain  affections  of  the 
anal  region  (Hartmann  and  Lieffring).  It  has  been  obtained  in  pure 
culture  from  abscesses  in  various  parts  of  the  body,  from  the  valves 
of  the  heart  in  endocarditis,  from  the  pleural  cavity  in  empyema,  etc. 
It  has  also  been  found  in  the  blood,  as  a  result  of  general  infection 
following  cystitis  and  pyelonephritis  (Sittmann  and  Barnow). 

Varieties. — Booker,  in  his  extended  studies  relating  to  the  bac- 
teria present  in  the  faeces  of  infants  suffering  from  summer  diarrhoea, 
has  isolated  seven  varieties  "  which  closely  resemble  Bacterium  coli 
commune  in  morphology  and  growth  in  agar,  neutral  gelatin,  and 
potato,  but  by  means  of  other  tests  a  distinction  can  be  made  between 
them." 

Some  of  the  pathogenic  bacteria  heretofore  described  are  also 
closely  allied  to  the  "  colon  bacillus  "  and  by  some  bacteriologists  are 
supposed  to  belong  to  the  same  group — i.e.,  to  be  varieties  of  the 
same  species  rather  than  independent  species  with  fixed  characters. 
Whatever  may  be  the  remote  relationship,  the  typhoid  group,  the  hog- 


534  PATHOGENIC   AEROBIC   BACILLI 

cholera  group,  the  Bacillus  typhi  murium  of  Loffler,  the  bacillus  of 
Laser,  the  Bacillus  enteritidis  of  Gartner,  and  other  similar  bacilli 
appear  to  be  differentiated  from  one  another  by  characters  which 
justify  their  description  under  separate  names.  Still  it  is  difficult  to 
fix  upon  any  one  of  these  characters  to  which  specific  value  can  be 
attached ;  and,  in  view  of  the  many  varieties  found  in  nature  or  pro- 
duced artificially  in  laboratory  experiments,  we  are  not  justified  in 
asserting  that  our  classification  of  these  low  organisms  has  any  sub- 
stantial scientific  foundation.  The  difficulties  attending  an  attempt 
to  establish  specific  characters  are  well  illustrated  by  the  extensive 
literature  relating  to  the  differentiation  of  bacilli  belonging  to  the 
typhoid  group  from  those  belonging  to  the  colon  group.  The  main 
points  upon  which  the  distinction  must  depend  have  been  referred  to 
in  the  section  devoted  to  the  typhoid  bacillus. 

Fremlin  (1893)  has  made  a  comparative  study  of  the  colon  bacil- 
lus from  various  sources.  He  finds  the  common  characters  of  gas 
production  in  media  containing  sugar  and  coagulation  of  milk.  Cul- 
tivated from  different  animals  the  morphology  is  the  same,  but  there 
are  differences  as  regards  motility.  The  most  active  movements  are 
said  to  be  exhibited  in  the  bacillus  from  man,  while  the  variety  ob- 
tained from  the  intestines  of  rabbits  showed  scarcely  any  movements. 
The  different  varieties  displayed  considerable  differences  in  their 
growth  upon  potato. 

Dreyfuss  (1894)  finds  decided  differences  in  the  pathogenic  viru- 
lence of  the  colon  bacillus  from  healthy  individuals  and  from  those 
suffering  from  various  intestinal  disorders.  A  culture  from  the  dis- 
charges of  a  fatal  case  of  cholera  nostras  proved  to  be  exceptionally 
virulent — tested  by  intraperitoneal  injections  in  guinea-pigs.  Gilbert 
(1895),  as  a  result  of  his  extended  researches,  concludes  that  there  are 
five  principal  types  among  the  bacilli  most  nearly  related  to  the  colon 
bacillus:  1st.  Bacilli  which  differ  from  the  colon  bacillus  by  their 
being  non-motile.  This  type  includes  two  varieties :  one  gives  thick 
yellowish  colonies  upon  gelatin  plates  and  numerous  gas  bubbles  on 
potato — this  is  the  bacille  lactique  of  Pasteur  and  the  Bacillus  lactis 
aerogenes  of  Escherich ;  the  other  gives  thin,  bluish-white  colonies 
and  includes  the  bacille  de  V  endocardite  of  Gilbert  and  Lion.  2d. 
Bacilli  which  differ  from  the  colon  bacillus  by  the  fact  that  cultures 
do  not  give  the  indol  reaction.  3d.  Bacilli  which  do  not  cause  the 
fermentation  of  lactose.  4th.  Bacilli  which  are  not  motile  and 
do  not  ferment  lactose.  5th.  Bacilli  which  are  not  motile,  do  not 
give  the  indol  reaction,  and  do  not  ferment  lactose. 

Theobald  Smith  (1895)  gives  the  following  account  of  his  method 
of  detecting  bacilli  of  the  "  colon  group  "  in  water  : 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  535 

4 '  The  method  followed  by  the  writer  in  the  general  bacteriological  exam- 
ination of  water  consists,  first,  in  the  preparation  of  gelatin  plates  for  the 
usual  enumeration  ;  and,  second,  in  the  addition  to  every  one  of  ten  fermen- 
tation tubes,  containing  a  oiie-per-cent  dextrose  bouillon,  a  certain  quantity 
of  water.  This  is  added  most  easily  by  first  diluting  the  water,  so  that  one  or 
two  cubic  centimetres  are  equivalent  to  the  quantity  which  it  is  desired  to  add 
to  each  tube.  Pipettes  graduated  by  drops  are  convenient,  but  not  so  accurate. 
In  case  of  ground  water  it  is  well  to  prepare  in  addition  a  flask  containing 
fifty  to  one  hundred  cubic  centimetres  of  the  water,  and  an  equal,  or  greater, 
quantity  of  bouillon,  to  which  sugar  is  not  added.  Plates  may  be  prepared 
from  this  flask  after  sixteen  to  twenty -four  hours.  When  gas  begins  to  ap- 
pear in  the  fermentation  tubes,  the  amount  accumulated  at  the  end  of  each 
twenty-four  hours  should  be  marked  with  a  glass  pencil  on  the  tube.  From 
these  tubes,  which  contain  fifty  to  sixty  per  cent  of  gas  on  the  third  day,  and 
are  very  strongly  acid,  plates  may  be  prepared  to  confirm  the  indications  of 
Bacillus  coli.  This,  however,  is  not  essential,  for  the  writer  has  found  as 
yet  no  species  having  these  fermentative  characters  which  is  not  one  of  the 
following  :  Bacillus  coli,  Bacillus  lactis  aerogenes,  Bacillus  enteriditis,  Bacil- 
lus typhi  murium,  Bacillus  cholera?  suis.  The  three  last-mentioned  species 
are  probably  as  rare  in  water  as  Bacillus  typhosus  itself. 

' '  My  own  experience  coincides  with  that  of  Matthews  when  he  states  that 
ninety-two  per  cent  of  all  bacteria  in  ground  water  are  suppressed  in  the 
thermostat.  While  the  addition  of  0.5  cubic  centimetre,  or  even  more,  of 
such  water  may  fail  to  produce  cloudiness  in  any  of  the  series  of  fermenta- 
tion tubes,  the  same  quantity,  or  less,  of  surface  water  never  fails  to  infect 
the  tubes." 

Bacillus  Coli  Communis  in  Peritonitis. — The  researches  of  A. 
Frankel  show  that  Bacillus  coli  communis  may  be  obtained  in  pure 
cultures  from  the  exudate  into  the  peritoneal  cavity  in  a  considerable 
proportion  of  the  cases  of  peritonitis,  and  there  is  good  reason  for 
believing  that  in  these  cases  it  was  the  cause  of  the  inflammatory 
process.  Thirty-one  cases  were  examined  by  Frankel,  with  the  fol- 
lowing result:  Pure  cultures  of  Bacillus  coli  communis  were  obtained 
in  nine  cases ;  of  Streptococcus  (pyogenes  ?)  in  seven ;  of  Bacillus 
lactis  aerogenes  in  two;  of  "  diplococcus  pneumonise "  in  one;  of 
Staphylococcus  pyogenes  aureus  in  one.  Of  the  remaining  eleven 
cases,  seven  gave  mixed  cultures,  and  in  three  of  these  Bacillus  coli 
communis  was  the  most  abundant  species.  The  author  referred  to 
has  also  shown  that  pure  cultures  of  Bacillus  coli  communis  injected 
into  the  cavity  of  the  abdomen  of  rabbits  cause  a  typical  peritonitis. 
The  present  writer  has  frequently  obtained  the  same  result  in  experi- 
ments made  with  this  bacillus.  It  would  appear,  therefore,  that  the 
peritonitis  which  so  constantly  results  from  wounds  of  the  intestine 
is  probably  due,  to  a  considerable  extent,  to  the  introduction  of  this 
microorganism  from  the  lumen  of  the  intestine,  where  it  is  con- 
stantly found,  into  the  peritoneal  cavity,  where  the  conditions  are 
favorable  for  its  rapid  development. 


536  PATHOGENIC   AEROBIC  BACILLI 


BACILLUS   LACTIS   AEROGENES. 

Obtained  by  Escherich  (1886)  from  the  contents  of  the  small  intestine  of 
children  and  animals  fed  upon  milk  ;  in  smaller  numbers  from  the  fogces  of 
milk-fed  children,  and  in  one  instance  from  uncooked  cow's  milk. 

Morphology. — Short  rods  with  rounded  ends,  from  1  to 
\  2  P  in  length  and  from  0.1  to  0.5  n  broad;   short  oval  and 

•j^   %*         spherical  forms  are  also  frequently  observed,  and,  under 
98         certain  circumstances,  longer  rods  — 3  v— may  be  developed : 
$*°  /  $         usually  united  in  pairs,  and  occasionally  in  chains  contain- 
ing several  elements.    In  some  of  the  larger  cells  Escherich 
has  observed  unstained  spaces,  but  was  not  able  to  obtain 
any  evidence  that  these  represent  spores. 

This  bacillus  stains  readily  with  the  ordinary  aniline 
colors,  but  does  not  retain  its  color  when  treated  by  Gram's 

ciicricii.)  .  i      i 

method. 

Biological  Characters. — An  aerobic  (facultative  anaerobic),  non-liquefy- 
ing, non  motile  bacillus.  Does  not  form  spores.  Grows  in  various  culture 
media  at  the  room  temperature — more  rapidly  in  the  incubating  oven. 
Upon  gelatin  plates,  at  the  end  of  twenty-four  hours,  small  white  colonies 
are  developed.  Upon  the  surface  these  form  hemispherical,  soft,  shining 
masses  which,  examined  under  the  microscope,  are  found  to  be  homogeneous 
and  opaque,  with  a  whitish  lustre  by  reflected  light.  The  deep  colonies  are 
spherical  and  opaque  and  attain  a  considerable  size.  In  gelatin  stab  cul- 
tures the  growth  resembles  that  of  Friedlander's  bacillus — i.e..  an  abundant 
growth  along  the  line  of  puncture  and  a  rounded  mass  upon  the  surface, 
forming  a  " nail-shaped"  growth.  In  old  cultures  the  upper  portion  of  the 
gelatin  is  sometimes  clouded,  and  numerous  gas  bubbles  may  form  in  the 
gelatin.  Upon  the  surface  of  nutrient  agar  an  abundant,  soft,  white  layer 
is  developed.  Upon  old  potatoes,  in  the  incubating  oven,  at  the  end  of 
twenty-four  hours  a  yellowish -white  layer,  several  millimetres  thick,  is 
developed,  which  is  of  paste-like  consistence  and  contains  about  the  peri- 
phery a  considerable  number  of  small  gas  bubbles ;  this  layer  increases  in 
dimensions,  has  an  irregular  outline,  and  larger  and  more  numerous  gas 
bubbles  are  developed  about  the  periphery,  some  the  size  of  a  pea;  later  the 
whole  surface  of  the  potato  is  covered  with  a  creamy,  semi-fluid  mass  filled 
with  gas  bubbles.  On  young  potatoes  the  development  is  different ;  a  rather 
luxuriant,  thick,  white  or  pale-yellow  layer  is  formed,  which  is  tolerably 
dry  and  has  irregular  margins;  the  surface  is  smooth  and  shining,  and  a 
few  minute  gas  bubbles  only  are  formed  after  several  days. 

Pathogenesis. — Injections  of  a  considerable  quantity  of  a  pure  culture 
into  the  circulation  of  rabbits  and  of  guinea-pigs  give  rise  to  a  fatal  result 
within  forty-eight  hours. 

In  his  first  publication  relating  to  "  the  bacteria  found  in  the  dejecta  of 
infants  afflicted  with  summer  diarrhoea,"  Booker  has  described  a  bacillus 
which  he  designates  by  the  letter  B,  which  closely  resembles  Bacillus  laclis 
aerogeiies  and  is  probably  identical  with  it.  He  says : 

"  Summary  of  Bacillus  B. — Found  nearly  constantly  in  cholera  infan- 
tum  and  catarrhal  enteritis,  and  generally  the  predominating  form.  It 
appeared  in  larger  quantities  in  the  more  serious  cases.  It  was  not  found 
in  the  dysenteric  or  healthy  faeces.  It  resembles  the  description  of  the  Ba- 
cillus lactis  aerogenes,  but  the  resemblance  does  not  appear  sufficient  to  con- 
stitute an  identity,  and,  in  the  absence  of  a  culture  of  the  latter  for  com- 
parison, it  is  considered  a  distinct  variety  for  the  following  reasons :  Bacillus 
B  is  uniformly  larger,  its  ends  are  not  so  sharply  rounded,  and  in  all  culture 
media  long,  thick  filaments  are  see  a,  and  many  of  the  bacilli  have  the  pro- 
toplasm gathered  in  the  centre,  leaving  the  poles  clear.  There  is  some 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS. 


537 


difference  in  their  colony  growth  on  gelatin,  and  in  gelatin  stab  cultures 
bacillus  B  does  not  show  the  nail- form  growth  with  marked  end  swelling  in 
the  depth.  In  potato  cultures  the  Bacillus  lactis  aerogenes  shows  a  differ- 
ence between  old  and  new  potatoes,  while  bacillus  B  does  not  show  any 
difference. 

"Bacillus  B  possesses  decided  pathogenic  properties,  which  was  shown 
both  by  hypodermic  injections  and  feeding  with  milk  cultures." 

BACILLUS   ACIDIFORMANS. 

Obtained  by  the  writer  (1888)  from  a  fragment  of  yellow-fever  liver  pre- 
served for  forty-eight  hours  in  an  antiseptic  wrapping;  since  obtained  from 


FIG.  148.  FIG.  149. 

Fro.  148.— Bacillus  aciiiformans,  from  a  potato  culture,  x  1,000.  From  a  photomicrograph* 
(Sternberg  ) 

Fio.  149. — Culture  of  Bacillus  acidiformans  in  nutrient  gelatin,  end  of  four  days  at  22°  C. 
From  a  photograph.  (Sternberg.) 

liver  preserved  in  the  same  way  from  two  comparative  autopsies — i.e.,  not 
cases  of  yellow  fever. 

Morphology. — A  short  bacillus  with  rounded  corners,  sometimes  short 
oval  in  form ;  from  1|  to  3  fJ-  in  length  and  about  1.2  u-  in  breadth ;  may  grow 
out  into  filaments  of  5  to  10  //,  or  more,  in  length ;  in  some  cultures  the  short 
oval  form,  predominates. 

Stains  readily  with  the  aniline  colors  usually  employed,  and  by  Gram's 
method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  Grows  rapidly  at 
the  room  temperature  in  the  usual  culture  media.  Grows  in  decidedly  acid 
media;  in  culture  media  containing  glycerin  or  glucose  it  produces  an  abun- 
dant evolution  of  carbon  dioxide,  and  a  volatile  acid  is  formed. 

It  does  not  liquefy  gelatin,  and  in  stab  cultures  grows  abundantly  both 
on  the  surface  and  along  the  line  of  puncture.  At  the  end  of  twenty-four 
hours,  at  22°  C.,  a  rounded  white  mass  is  formed  upon  the  surface,  resembling 
the  growth  of  Friedlander's  bacillus ;  at  the  bottom  of  the  line  of  puncture 
the  separate  colonies  are  spherical,  opaque,  and  pearl-like  by  reflected  light. 
Gas  bubbles  are  formed  in  the  gelatin.  At  the  end  of  a  week  the  surface  is 
covered  with  a  thick,  white,  semi-fluid  mass. 

In  gelatin  roll  tubes  the  superficial  colonies  are  translucent  or  opaque, 
and  circular  or  somewhat  irregular  in  outline;  by  reflected  light  they  are 


538 


PATHOGENIC   AEROBIC  BACILLI 


.slightly  iridescent;  the  deep  colonies  are  spherical,  opaque,  and  homo- 
geneous. 

The  growth  upon  the  surface  of  nutrient  agar  is  abundant  and  rapid,  of 
a  shining  milk-white  color,  and  cream-like  in  consistence.  An  abundant 
development  forms  alon^  the  line  of  puncture  and  the  culture  medium  is 
split  up  by  gas  bubbles.  In  glycerin-agar  the  evolution  of  gas  is  very  abun- 
dant and  the  culture  medium  acquires  an  intensely  acid  reaction. 

On  potato  the  growth  is  abundant  and  rapid  at  a  temperature  of  20°  to 
30°  C.,  forming  a  thick,  semi-fluid  mass  of  a  milk-white  color. 

I  have  not  obtained  any  evidence  that  this  bacillus  forms  spores;  the 
cultures  are  sterilized  by  ten  minutes'  exposure  to  a  temperature  of  160°  F. 

When  cultivated  in  bouillon  to  which  five  per  cent  of  glycerin  has  been 
added  the  culture  medium  acquires  a  milky  opacity,  and  there  is  a  copious 
precipitate,  of  a  viscid  consistence,  consisting  of  bacilli ;  during  the  period 
of  active  development  the  surface  is  covered  with  gas  bubbles,  as  in  a  sac- 
charine liquid  undergoing  alcoholic  fermentation,  and  the  liquid  has  a  de- 
cidedly acid  reaction. 

Pathogenesis. — Pathogenic  for  rabbits  and  for  guinea  pigs  when  injected 
into  the  cavity  of  the  abdomen — one  to  two  cubic  centimetres  of  a  culture  in 
bouillon.  The  animal  usually  dies  in  less  than  twenty-four  hours.  The 
bacilli  are  found  in  the  blood  in  rather  small  numbers,  and  are  frequently 
seen  in  the  interior  of  the  leucocytes.  The  spleen  is  enlarged,  the  liver 
normal,  the  intestine  usually  hyperaemic. 


BACILLUS   CUNICULICIDA   HAVANIENSIS. 

Obtained  by  the  writer  (1889)  from  the  contents  of  the  intestine  of  yellow- 
fever  cadavers,  and  also  from  fragments  of  yellow-fever  liver  preserved  for 

forty-eight  hours  in  an  antiseptic  wrap- 
ping—my bacillus  x,  Havana,  1889. 

Morphology. — This  bacillus  resembles 
the  colon  bacillus  in  form,  but  is  some- 
what larger,  from  2  to  4  //  in  length  and 
from  0.8  to  1  JJ>  in  diameter  ;  sometimes 
associated  in  pairs ;  may  grow  out  into 
short  filaments — not  common.  The  ends 
of  the  rods  are  rounded,  and  under  cer- 
tain circumstances  vacuoles  are  seen  at 
the  extremities,  especially  in  potato  cul- 
tures. 

Stains  quickly  with  the  aniline  colors 
usually  employed,  and  also  by  Gram's 
method. 

Biological  Characters. — An  aerobic 
and  facultative  anaerobic,  non-lique- 
fying bacillus.  Under  certain  circum- 
stances may  exhibit  active  movements, 
but  is  usually  motionless. 

A  very  curious  thing  with  reference 
to  this  bacillus  is  that  it  presented  ac- 
tive movements  in  my  cultures  made  directly  from  yellow-fever  cadavers, 
but  that  these  movements  were  not  constant,  and  that  since  my  return  to 
Baltimore  I  have  not,  as  a  rule,  observed  active  movements  in  cultures  from 
the  same  stock,  which,  however,  preserved  their  pathogenic  power  and  other 
characters.  In  Havana  these  movements  were  usually  not  observed  in  all 
the  bacilli  in  a  field  under  observation,  but  one  and  another  would  start  from 
a  quiescent  condition  on  an  active  and  erratic  course ;  sometimes  spinning 
actively  upon  its  axis,  and  again  shooting  across  the  field  as  if  propelled  by 
a  nagellum. 


FIG.  150.— Bacillus  cuniculicida  Havani- 
•ensis,  from  a  single  colony  in  nutrient  gela- 
tin, x  1,000.  From  a  photomicrograph. 
(Sternberg.) 


NOT   DESCRIBED   IN  PREVIOUS  SECTIONS. 


539 


My  notes  indicate  that  cultures  passed  through  the  guinea-pig  are  more 
apt  to  be  motile. 

In  gelatin  stab  cultures  the  growth  of  bacillus  x  resembles  that  of  the 
colon  bacillus,  but  the  colonies  at  the  bottom  of  the  line  of  puncture  are 
more  opaque  and  not  of  a  clear  amber  color  like  that  of  colonies  of  the  colon 
bacillus.  Upon  the  surface  the  growth  is  thicker  than  that  of  the  colon 
bacillus,  and  forms  a  milk-white,  soft  mass. 

The  colonies  in  gelatin  Esmarch  roll  tubes  vary  considerably  at  different 
times.  Deep  colonies  are  usually  spherical,  homogeneous,  light  brown  in 
color,  and  more  opaque  than  the  similar  colonies  of  the  colon  bacillus.  At 
the  end  of  a  few  days  the  deep  colonies  become  quite  opaque,  and  may  be 
Jobate,  like  a  mulberry,  or  coarsely  granular;  sometimes  the  aeep  colonies 
.have  an  opaque  central  portion  surrounded  by  a  transparent  marginal  zone. 

In  old  gelatin  roll  tubes  these  deep  colonies  form  opaque  white  hemi- 


FIG.  151.  FIG.  153. 

Fig.  151.— Bacillus  cuniculicida  Havaniensis;  colonies  in  gelatin  roll  tube,  third  day  at  20°  C. 
X  6.  From  a  photograph.  (Sternberg.) 

Fio.  152. — Bacillus  cuniculicida  Havaniensis ;  colonies  in  gelatin  roll  tube,  end  of  forty-eight 
hours.  X  10.  From  a  photograph.  (Sternberg.) 


spheres  projecting  from  the  surface  of  the  dried  culture  medium,  and  little 
tufts  of  acicular  crystals  are  sometimes  observed  to  project  from  the  side  of 
such  old  colonies. 

The  superficial  colonies  are  circular  or  irregular  in  outline,  with  trans- 
parent margins  a.nd  an  opaque  central  portion,  sometimes  corrugated.  They 
are  finely  granular  and  iridescent  by  reflected  light,  and  of  a  milk-white 
color ;  by  transmitted  light  they  have  a  brownish  color.  Young  colonies 
closely  resemble  those  of  the  colon  bacillus.  This  bacillus  grows  well  at  a 
temperature  of  20°  C.  (68°  F.),  but  more  rapidly  and  luxuriantly  at  a  higher 
temperature — 30°  to  35°  C. 

It  grows  well  in  agar  cultures,  and  especially  in  glycerin-agar,  in  which 
it  produces  some  gas  and  an  acid  reaction.  The  growth  on  the  surface 
of  glycerin-agar  cultures  is  white,  cream-like  in  consistence,  and  quite  abun- 
dant. 

It  grows  well  in  an  agar  or  gelatin  medium  made  acid  by  the  addition  of 
'0.2  per  cent  (1:  500)  of  hydrochloric  acid. 

In  cocoanut  water  it  multiplies  rapidly,  producing  a  milky  opacity  of  the 
previously  transparent  fluid,  an  acid  reaction,  and  an  evolution  of  carbon 
dioxide. 

On  potato  it  produces  a  thick  layer,  which  may  cover  the  entire  surface 
in  three  or  four  days,  and  which  has  a  dirty-white,  cream- white,  or  pinkish- 


540  PATHOGENIC   AEROBIC   BACILLI 

white  color  and  cream-like  consistence.     The  growth  upon  potato  varies  at 
different  times,  evidently  owing1  to  differences  in  the  potato. 

When  stained  preparations  are  examined  with  the  full  light  of  the  Abbe 
condenser  the  ends  of  some  of  the  rods  appear  to  be  cut  away,  leaving-  a  con- 
cave extremity ;  but  by  using-  a  small  diaphragm  to  obtain  definition  it  will 
be  seen  that  the  cell  wall  extends  beyond  the  stained  portion  of  the  rod  and 
includes  what  appears  to  be  a  vacuole.  There  is  no  reason  to  believe  that 
this  appearance  is  due  to  the  presence  of  an  end  spore,  for  the  supposed 
vacuole  is  not  refractive,  as  a  spore  would  be,  and  my  experiments  on  the 
thermal  death-point  of  this  bacillus  indicate  that  it  does  not  form  spores. 
Cultures  are  sterilized  by  exposure  for  ten  minutes  to  a  temperature  of  160° 
F.  (71.2°  C.). 

Pathogenesis. — Very  pathogenic  for  rabbits  when  injected  into  the  cavity 
of  the  abdomen.  Injections  of  a  small  quantity  of  a  pure  culture  into  the 
ear  vein  or  subcutaneously  generally  give  a  negative  result.  Injections  of 
from  one  to  five  cubic  centimetres  of  a  culture  in  bouillon,  blood  serum,  or 
agua  coco,  into  the  cavity  of  the  abdomen,  frequently  prove  fatal  to  rabbits 
in  a  few  hours — two  to  six. 

The  negative  results  obtained  in  injecting  cultures  beneath  the  skin  or 
into  the  ear  vein  of  rabbits  show  that  this  bacillus  does  not  induce  a  fatal 
septicaemia  in  these  animals,  and  the  fatal  result  when  injections  are  made 
into  the  peritoneal  cavity  does  not  appear  to  be  due  to  an  invasion  of  the 
blood,  but  rather  to  the  local  effect  upon  the  peritoneum,  together  with  the 
toxic  action  of  the  chemical  products  resulting  from  its  growth. 

It  is  true  that  I  have  always  been  able  to  recover  the  bacillus  from  the 
liver,  or  from  blood  obtained  from  one  of  the  cavities  of  the  heart,  even  in 
animals  which  succumb  within  a  few  hours  to  an  injection  made  into  the 
cavity  of  the  abdomen.  But  the  direct  examination  of  the  blood  shows  that 
the  bacilli  are  present  in  very  small  numbers,  and  leads  me  to  believe  that 
the  bacillus  does  not  multiply,  to  any  considerable  extent  at  least,  in  the 
circulating  fluid. 

The  spleen  is  not  enlarged,  as  is  the  case  in  anthrax,  rabbit  septicaemia, 
and  other  diseases  in  which  the  pathogenic  microorganism  multiplies  abun- 
dantly in  the  blood. 

On  the  other  hand,  there  is  evidence  of  local  inflammation  in  the  peri- 
toneal cavity.  When  death  occurs  within  a  few  hours  the  peritoneum  is 
more  or  less  hyperaemic  and  there  is  a  considerable  quantity  of  straw-colored 
fluid  in  the  cavity  of  the  abdomen.  When  the  animal  lives  for  twenty 
hours  or  more  there  is  a  decided  peritonitis  with  a  fibrinous  exudation  upon 
the  surface  of  the  liver  and  intestine.  Usually  the  liver,  in  animals  which 
die  within  twenty-four  hours,  is  full  of  blood,  rather  soft,  and  dark  in  color. 
In  a  single  instance  I  found  the  liver  to  be  of  a  light  color  and  loaded  with 
fat. 

The  rapidly  fatal  effect  in  those  cases  in  which  I  have  injected  two  or 
more  cubic  centimetres  of  a  culture  into  the  cavity  of  the  abdomen  has  led 
me  to  suppose  that  death  results  from  the  toxic  effects  of  a  ptomaine  con- 
tained in  the  culture  at  the  time  of  injection.  The  symptoms  also  give  sup- 
port to  this  supposition.  The  animal  quickly  becomes  feeble  and  indisposed 
to  move,  and  some  time  before  death  lies  helpless  upon  its  side,  breathing 
regularly,  but  is  too  feeble  to  get  up  on  its  feet  when  disturbed.  Death  some- 
times occurs  in  convulsions,  but  more  frequently  without— apparently  from 
heart  failure. 

Pathogenic  also  for  guinea-pigs  when  injected  into  the  cavity  of  the- 
abdomen,  but  death  does  not  occur  in  so  short  a  time — eighteen  to  twenty 
hours.  The  comparative  researches  of  Reed  and  Carroll  indicate  that  this  is- 
a  pathogenic  variety  of  the  colon  bacillus. 


NOT   DESCRIBED   IN  PREVIOUS   SECTIONS.  541 


BACILLUS   LEPORIS  LETHALIS. 

Obtained  by  Dr.  Paul  Gibier  (1888)  from  the  contents  of  the  intestine  of 
yellow-fever  patients;  also  by  the  writer  from  the  same  source  (1888,  1889) 
in  exceptional  cases  and  in  comparatively  small  numbers.  Named  and  de- 
scribed by  present  writer. 

Morphology. — Bacilli  with  rounded  ends,  from  1  to  3  ^  in  length  and 
about  0.5  H  in  breadth.  The  length  may  vary  in  the  same  culture  from  a 
short  oval  to  rods  which  are  two  or  three  times  as  long  as  broad,  or  it  may 
grow  out  into  flexible  filaments  of  considerable  length.  In  recent  cultures 
the  bacilli  are  frequently  united  in  pairs. 

Stains  readily  with  the  aniline  colors  usually  employed.  In  cultures 
which  are  several  days  old,  or  in  recent  cultures  when  the  stained  prepara- 
tion is  washed  in  alcohol,  the  ends  of  the  rods  are  commonly  more  deeply 
stained  than  the  central  portion — "  end  staining";  and  in  old  cultures  some 
of  the  bacilli  are  very  faintly  stained. 

Biological  Characters. — A.U  aerobic,  liquefying,  actively  motile  bacillus. 
Does  not  form  spores. 

In  gelatin  stab  cultures,  at  the  end  of  twenty-four  hours  at  a  tempe- 
rature of  20°  to  22°  C.,  there  is  an  abundant  development  along-  the  line  of 
puncture  and  commencing  liquefaction  at  the  surface.  Later  the  liquefaction 
is  funnel-shaped,  and  there  is  an  opaque  white  central  core  along  the  line 
of  puncture,  with  liquefied  gelatin  around  it.  Liquefaction  progresses  most 
rapidly  at  the  surface,  and  in  the  course  of  three  or  four  days  the  upper  por- 
tion of  the  gelatin  for  a  distance  of  half  an  inch  or  more  is  completely  lique- 
fied, and  an  opaque  white  mass,  composed  of  bacilli,  rests  upon  the  surface 
of  the  unliquefied  portion. 

In  gelatin  roll  tubes  the  young  colonies  upon  the  surface  are  transparent 
and  resemble  somewhat  small  fragments  of  broken  glass ;  later  liquefaction 
occurs  rapidly.  Deep  colonies  in  gelatin  roll  tubes,  or  at  the  bottom  of  stick 
cultures,  are  spherical,  translucent,  and  of  a  pale  straw  color. 

Upon  the  surface  of  nutrient  agar  it  grows  rapidly,  forming  a  rather  thin, 
translucent,  shining,  white  layer,  which  covers  the  entire  surface  at  the  end 
of  two  or  three  days  at  a  temperature  of  20°  C. 

Upon  potato  the  growth  is  rapid  and  thin,  covering  the  entire  surface, 
and  is  of  a  pale-yellow  color. 

This  bacillus  grows  at  a  comparatively  low  temperature,  and  its  vitality 
is  not  destroyed  by  exposure  for  an  hour  and  a  half  in  a  freezing  mixture  at 
15°  C.  below  zero  (5°  F.). 

Decided  growth  occurred  in  a  stick  culture  in  gelatin  exposed  in  Balti- 
more during  the  month  of  January  in  an  attic  room.  During  the  twenty- 
two  days  of  exposure  the  highest  temperature,  taken  at  9  A.M.  each  day, 
was  11°  C.,  and  the  lowest  2°  C.  At  a  temperature  of  16°  to  20°  C.  develop- 
ment in  a  favorable  culture  medium  is  rapid. 

There  is  no  evidence  that  this  bacillus  forms  spores ;  cultures  are  sterilized 
by  exposure  to  a  temperature  of  60°  C.  for  ten  minutes. 

Coagulated  blood  serum  is  liquefied  by  this  bacillus.  It  retains  its  vitality 
for  a  long  time  in  old  cultures,  having  grown  freely  when  replanted  at  the 
end  of  a  year  from  a  hermetically  sealed  tube  containing  a  pure  culture  in 
blood  serum. 

Pathogenesis. — This  bacillus  is  very  pathogenic  for  rabbits  when  injected 
into  the  cavity  of  the  abdomen  in  quantities  of  one  cubic  centimetre  or  more ; 
it  is  less  pathogenic  for  guinea-pigs,  and  is  not  pathogenic  for  white  rats 
when  injected  subcutaneously.  Gelatin  cultures  seem  to  possess  more  in- 
tense pathogenic  power  than  bouillon  cultures,  and  cultures  from  the  blood 
of  an  animal  recently  dead  as  the  result  of  an  inoculation  are  more  potent 
than  those  from  my  original  stock  which  had  not  been  passed  through  a 
susceptible  animal. 


542  PATHOGENIC   AEROBIC  BACILLI 

The  mode  of  death  in  rabbits  is  quite  characteristic.  A  couple  of  hours 
after  receiving  in  the  cavity  of  the  abdomen  two  or  three  cubic  centimetres 
of  a  liquefied  gelatin  culture  the  animal  becomes  quiet  and  indisposed  to  eat 
or  move  about.  Soon  after  it  becomes  somnolent,  the  head  drooping  for- 
ward and  after  a  time  resting  between  the  front  legs,  with  the  nose  on  the 
floor  of  its  cage.  It  can  be  roused  from  this  condition,  and  raises  its  head  in 
an  indifferent  and  stupid  way  when  pushed  or  shaken,  but  soon  drops  off 
again  into  a  profound  sleep.  Frequently  the  animals  die  in  a  sitting  posi- 
tion, with  their  nose  resting  upon  the  floor  of  the  cage  between  the  front 
legs.  I  have  not  seen  this  lethargic  condition  produced  by  inoculations  with 
any  other  microorganism.  Convulsions  sometimes  occur  at  the  moment  of 
death. 

The  time  of  death  depends  upon  the  potency  of  the  culture  and  its  quan- 
tity as  compared  with  the  size  of  the  animal.  From  three  to  four  cubic 
centimetres  of  a  liquefied  gelatin  culture  usually  kill  a  rabbit  in  from  three 
to  seven  hours. 

The  rapidity  with  which  death  occurs  when  a  considerable  quantity  of  a 
liquefied  gelatin  culture  is  injected  into  the  cavity  of  the  abdomen,  and  the 
somnolence  which  precedes  death,  give  rise  to  the  supposition  that  tV'e  lethal 
effect  is  due  to  the  presence  of  a  toxic  chemical  substance  rather  than  to  a 
multiplication  of  the  bacillus  in  the  body  of  the  animal.  And  this  view  is 
supported  by  the  fact  that  animals  frequently  recover  when  the  dose  admin- 
istered is  comparatively  small  and  especially  when  it  is  injected  subcuta- 
neously. 

In  all  cases  in  which  death  occurs,  even  when  but  a  few  hours  have 
elapsed  since  the  inoculation  was  made,  I  have  recovered  the  bacillus  in 
cultures  made  from  blood  obtained  from  the  heart  or  the  interior  of  the 
liver,  and,  as  stated,  these  cultures  appear  to  have  a  greater  virulence  than 
those  not  passed  through  the  rabbit. 

In  sections  of  the  liver  and  kidney  stained  with  Loffler's  solution  of 
methylene  blue  the  bacilli  are  seen,  and  are  often  in  rather  long- jointed  fil- 
aments. 

BACILLUS   PYOCYANEUS. 

Synonyms. — Bacillus  of  green  pus  ;  Microbe  du  pus  bleu;  Bacil- 
len  des  griinblauen  Eiters ;  Bacterium  aeruginosum. 

Obtained  by  Gessard  (1882)  from  pus  having  a  green  or  blue 
color,  and  since  carefully  studied  by  Gessard,  Charrin,  and  others. 

This  bacillus  appears  to  be  a  widely  distributed 
saprophyte,  which  is  found  occasionally  in  the 
purulent  discharges  from  open  wounds,  and  some- 
times in  perspiration  and  serous  wound  secretions 
(Gessard) .    The  writer  obtained  it,  in  one  instance, 
FIG.  153.  —  Bacillus      in  cultures  from  the  liver  of  a  yellow-fever  cada- 
X  7°°'       ver  (Havana,  1888). 

Morphology. — A  slender  bacillus  with  rounded 
ends,  somewhat  thicker  than  the  Bacillus  murisepticus  and  of  about 
the  same  length  (Fliigge) ;  frequently  united  in  pairs,  or  chains  of  four 
to  six  elements;  occasionally  grows  out  into  filaments. 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacil- 
lus. Grows  readily  in  various  culture  media  at  the  room  tempera- 
ture— more  rapidly  in  the  incubating  oven.  Is  a  facultative  anae- 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS.  543- 

robic  (Frankel).  Does  not  form  spores.  The  thermal  death-point, 
as  determined  by  the  writer,  is  56°  C.,  the  time  of  exposure  being  ten 
minutes.  In  gelatin  plate  cultures  colonies  are  quickly  developed, 
which  give  to  the  medium  a  fluorescent  green  color  ;  at  the  end  of 
two  or  three  days  liquefaction  commences  around  each  colony,  and 
usually  the  gelatin  is  completely  liquefied  by  the  fifth  day.  Before 
liquefaction  commences  the  deep  colonies,  under  a  low  power,  appear 
as  spherical,  granular  masses,  with  a  serrated  margin,  and  have  a 
yellowish-green  color ;  the  superficial  colonies  are  quite  thin  and 
finely  granular  ;  at  the  centre,  where  they  are  thickest,  they  have  a 
greenish  color,  which  fades  out  towards  the  periphery. 

In  stab  cultures  in  nutrient  gelatin  development  is  most  abun- 
dant near  the  surface,  and  causes  at  first  liquefaction  in  the  form 
of  a  shallow  funnel ;  later  the  liquefied  gelatin  is  separated  from 
that  which  is  not  liquefied  by  a  horizontal  plane,  and  a  viscid,  yel- 
lowish-white mass,  composed  of  bacilli,  accumulates  upon  this  sur- 
face, which  gradually  has  a  lower  level  as  liquefaction  progresses  ;. 
the  transparent,  liquefied  gelatin  above  is  covered  with  a  delicate, 
yellowish-green  film,  and  the  entire  medium  has  a  fluorescent  green 
color.  Upon  nutrient  agar  a  rather  abundant,  moist,  greenish- white 
layer  is  developed,  and  the  medium  acquires  a  bright  green-color, 
which  subsequently  changes  to  olive  green.  Upon  potato  a  viscid 
or  rather  dry,  yellowish-green  or  brown  layer  is  formed,  and  the 
potato  beneath  and  immediately  around  the  growth  has  a  green  color 
when  freely  exposed  to  the  air  or  to  the  vapors  of  ammonia.  In  milk 
the  casein  is  first  precipitated  and  then  gradually  dissolved,  while  at 
the  same  time  ammonia  is  developed.  The  green  pigment  is  formed 
only  in  the  presence  of  oxygen;  it  is  soluble  in  chloroform  and  may 
be  obtained  from  a  pure  solution  in  long,  blue  needles  ;  acids  change 
the  blue  color  to  red,  and  reducing  substances  to  yellow.  According 
to  Ledderhose,  it  is  an  aromatic  compound  resembling  anthracene, 
and  is  not  toxic.  According  to  Gessard's  latest  researches  (1890),  two 
different  pigments  are  produced  by  this  bacillus,  one  of  a  fluorescent 
green  and  the  other — pyocyanin — of  a  blue  color.  Cultivated  in  egg 
albumin  the  fluorescent  green  pigment,  which  changes  to  brown 
with  time,  is  alone  produced.  In  bouillon  and  in  media  containing 
peptone  or  gelatin  both  pigments  are  formed,  and  the  pyocyanin 
may  be  obtained  separately  by  dissolving  it  in  chloroform.  In  an 
alkaline  solution  of  peptone  (two  per  cent)  to  which  five  per  cent  of 
glycerin  has  been  added  the  blue  pigment  alone  is  formed. 

Pathogenesis. — The  experiments  of  Ledderhose,  Bouchard,  and 
others  show  that  this  bacillus  is  pathogenic  for  guinea-pigs  and  rab- 
bits. Subcutaneous  or  intraperitoneal  injections  of  recent  cultures — 


544  PATHOGENIC   AEROBIC   BACILLI 

one  cubic  centimetre  or  more  of  a  culture  in  bouillon — usually  cause 
the  death  of  the  animal  in  from  twelve  to  thirty-six  hours.  An  ex- 
tensive inflammatory  oedema  and  purulent  infiltration  of  the  tissues 
result  from  subcutaneous  inoculations,  and  a  sero-fibrinous  or  puru- 
lent peritonitis  is  induced  by  the  introduction  of  the  bacillus  into  the 
peritoneal  cavity.  The  bacillus  is  found  in  the  serous  or  purulent 
fluid  in  the  subcutaneous  tissues  or  abdominal  cavity,  and  also  in  the 
blood  and  various  organs,  from  which  it  can  be  recovered  in  pure 
cultures,  although  not  present  in  great  numbers,  as  is  the  case  in 
the  various  forms  of  septicaemia  heretofore  described.  When  smaller 
amounts  are  injected  subcutaneously  the  animal  usually  recovers 
after  the  formation  of  a  local  abscess,  and  it  is  subsequently  immune 
when  inoculated  with  doses  which  would  be  fatal  to  an  unprotected 
animal.  Immunity  may  also  be  secured  by  the  injection  of  a  con- 
siderable quantity  of  a  sterilized  culture.  Bouchard  has  also  pro- 
duced immunity  in  rabbits  by  injecting  into  them  the  filtered  urine 
of  other  rabbits  which  had  been  inoculated  with  a  virulent  culture  of 
the  bacillus.  It  has  been  shown  by  Bouchard,  and  by  Charrin  and 
Guignard,  that  in  rabbits  which  have  been  inoculated  with  a  culture 
of  the  anthrax  bacillus  a  fatal  result  may  be  prevented  by  soon  after 
inoculating  the  same  animals  with  a  pure  culture  of  the  Bacillus 
pyocyaneus.  The  experiments  of  Woodhead  and  Wood  indicate  that 
the  antidotal  effect  is  due  to  chemical  products  of  the  growth  of  the 
bacillus,  and  not  to  an  antagonism  of  the  living  bacterial  cells.  They 
were  able  to  obtain  similar  results  by  the  injection  of  sterilized  cul- 
tures of  Bacillus  pyocyaneus,  made  soon  after  infection  with  the 
anthrax  bacillus. 

Schimmelbusch  (1894)  reports  that  in  researches  made  by  Muh- 
sam  this  bacillus  was  found  in  the  axilla,  the  anal  region,  or  the  in- 
guinal fold  in  fifty  per  cent  of  the  healthy  individuals  examined. 
Its  presence  in  wounds  greatly  delays  the  process  of  repair  and  may 
give  rise  to  a  general  depression  of  the  vital  powers  from  the  ab- 
sorption of  its  toxic  products.  Schimmelbush  states  that  a  physician 
injected  0.5  cubic  centimetre  of  sterilized  (by  heat)  culture  into  his 
forearm.  That  as  a  result  of  this  injection,  after  a  few  hours  he  had 
a  slight  chill,  followed  by  fever,  which  at  the  end  of  twelve  hours 
reached  38.8°  ;  an  erysipelatous  -  like  swelling  of  the  forearm  oc- 
curred, and  the  glands  in  the  axilla  were  swollen  and  painful.  Re- 
covery occurred  without  the  formation  of  an  abscess.  Buchner  has 
related  a  similar  case. 

Krannhals  (1894)  refers  to  seven  cases  in  which  a  general  pyocy- 
aneus infection  in  man  was  found,  and  adds  an  eighth  from  his  own 
experience.  In  this  the  Bacillus  pyocyaneus  was  obtained,  post  mor- 


NOT  DESCRIBED   IN  PREVIOUS   SECTIONS.  545 

tern,  from  green  pus  in  the  pleural  cavity,  from  serum  in  the  peri- 
cardial sac,  and  from  the  spleen,  in  pure  culture. 

Martha,  Gruber,  Maggiora,  Gradenigo,  Kossel,  and  Rohrer  have 
reported  cases  in  which  the  Bacillus  pyocyaneus  has  been  obtained  in 
pure  cultures  from  pus  obtained  from  the  tympanic  cavity  in  middle- 
ear  disease.  Kossel  (1894)  relates  several  cases  in  his  own  experience 
which  led  him  to  the  conclusion  that,  in  children,  the  Bacillus  pyocy- 
aneus, through  general  blood  infection  or  indirectly  through  the 
absorption  of  its  toxic  products,  may  be  the  cause  of  death. 

The  following  varieties  of  this  bacillus  have  been  described  by 
bacteriologists: 

BACILLUS   PYOCYANEUS   /?   (P.    Ernst). 

Found  in  pus  from  bandages  colored  green. 

Morphology.  — Slender  bacilli  from  2  to  4  /-i  long — occasionally  5  to  6  fj- — 
and  from  0.5  to  0.75  Abroad;  sometimes  united  in  pairs,  or  chains  of  three 
elements. 

Biological  Characters. — An  aerobic,  liquefying,  actively  motile,  chro- 
mogenic  bacillus.  Produces  a  yellowish-green  pigment;  when  old  cul- 
tures are  shaken  up  with  chloroform  and  this  is  allowed  to  stand,  three 
layers  are  formed — an  upper,  clouded,  dirty -yellow  layer  ;  below  this  is  a 
milky,  pale-green  layer  ;  and  at  the  bottom  a  transparent,  azure-blue  layer. 
Spore  formation  has  not  been  demonstrated.  Grows  in  the  usual  culture 
media  at  the  room  temperature — more  rapidly  at  35°  C.  Upon  gelatin  plates 
colonies  are  formed  resembling  those  of  the  well-known  Bacillus  pyocyaneus, 
but  liquefaction  is  more  rapid.  In  gelatin  stick  cultures  funnel-shaped 
liquefaction  occurs  at  the  upper  part  of  the  line  of  puncture  by  the  third 
day,  and  progresses  more  rapidly  than  is  the  case  with  Bacillus  pyocyaneus 
under  the  same  circumstances  ;  on  the  fifth  day  a  bluish-green  color  is  de- 
veloped; by  the  twelfth  day  liquefaction  has  obliterated  the  entire  line  of 
growth  and  extends  to  the  margins  of  the  tube  ;  the  liquefied  gelatin  for  a 
depth  of  about  one  centimetre  has  a  dark  emerald-green  color,  and  a  film 
consisting  of  bacilli  is  seen  upon  the  surface.  Upon  the  surface  of  agar  a 
flat,  greenish- white,  dry  layer  is  formed  along  the  line  of  inoculation,  and 
the  agar  around,  at  the  end  of  a  week,  acquires  a  bluish-green  color.  Upon 
potato,  at  the  end  of  three  days,  an  abundant  dry  layer  of  a  fawn-brown 
color  has  developed  ;  this  is  surrounded  by  a  pale-green  coloration  of  the 
potato,  and  at  points  where  the  surface  is  fissured,  an  intense  dark-green 
color  is  developed;  the  growth  on  potato  has  a  more  or  less  wrinkled  appear- 
ance ;  when  one  of  the  fawn-colored  colonies  is  touched  with  the  platinum 
needle,  the  point  touched,  at  the  end  of  two  to  five  minutes,  acquires  an  in- 
tense dark  leaf -green  color,  which  reaches  its  maximum  intensity  in  about 
ten  minutes,  and  has  faded  out  again  at  the  end  of  half  an  hour.  Ernst  con- 
siders this  "chameleon  phenomenon"  the  most  characteristic  distinction 
between  the  bacillus  under  consideration  and  Bacillus  pyocyaneus.  In  milk 
a  green  color  is  developed  at  the  surface,  the  casein  is  precipitated  and  sub- 
sequently peptonized. 

Bacillus  pyocyaneus  pericarditidis.  Found  by  H.  C.  Ernst  in 
fluid  obtained  by  tapping  the  pericardial  sac  of  a  man  aged  forty- 
seven  years.  Fluid  was  drawn  from  the  pericardial  sac  on  four  dif- 
ferent occasions.  The  man  subsequently  "eloped."  Ernst  gives  the 
following  description  of  this  bacillus : 
35 


546  PATHOGENIC   AEROBIC  BACILLI 

ORIGIN. — Pericardial  fluid,  containing  also  bacilli  of  tuberculosis. 

FORM  AND  ARRANGEMENT. — Small  straight  bacilli,  with  rounded  ends, 
three  or  four  times  as  long  as  broad,  and  on  most  media  slightly  larger  than 
the  Bacillus  pyocyaneus  of  Gessard,  occurring  within  the  cells  in  the  origi- 
nal fluid,  and  sometimes  showing  two  or  three  end  to  end,  but  never  observed 
in  long  chains. 

MOTILITY. — Actively  motile  in  hanging-drop  culture.  No  cilia  or  flagel- 
la  have  been  demonstrated. 

GROWTH — Gelatin  :  Plates. — Colonies  appear  at  the  end  of  thirty-six  to 
forty-eight  hours  as  fine  white  points  in  the  interior,  and  upon  the  surface  of 
the  medium ;  edges  are  sharply  defined  ;  soon  there  appears  a  circular  zone 
of  liquefaction,  finally  passing  through  the  stratum  of  the  medium  with 
the  colony  at  the  bottom.  Under  a  low  power  the  centre  of  the  colony  may 
be  of  a  brownish  color.  On  the  second  day  a  greenish  tinge  may  be  seen 
about  the  individual  colonies  on  the  surface  which  spreads  through  the 
entire  medium.  The  plates  may  always  be  distinguished  from  those  of  the 
Bacillus  pyocyaneus  of  Gessard  by  the  bluish-green  when  contrasted  with 
the  yellowish-green  color  of  this  latter. 

Gelatin:  Needle  Cultures. — At  the  end  of  twenty-four  hours  a  small, 
saucer-shaped  depression  of  liquefaction  at  upper  end  of  needle  track,  which 
gradually  spreads  and  deepens  until  the  liquefaction  extends  straight  across 
the  tube,  and  about  half-way  down  the  needle  track.  A  bluish-green  fluores- 
cence appears  about  the  liquefied  portion  at  the  very  upper  part  of  the  gela- 
tin, later  changing  into  a  yellowish  green.  The  colony  is  deposited  as  a 
yellowish,  heavy  sediment  at  the  bottom  of  the  liquefied  portion,  the  upper 
part  of  which  is  clear.  A  small,  whitish  growth  occurs  along  the  remainder 
of  the  needle  track.  Old  cultures,  in  which  a  certain  amount  of  evapora- 
tion has  occurred,  assume  a  very  dark  greenish-black  color. 

Agar-agar. — Along  the  needle  track  appears  a  flat,  dry  colony  of  a  dirty 
grayish- white  color  spreading  out  upon  each  side  of  the  needle  track  and 
growing  at  first  upon  the  surface  of  the  water  of  condensation,  later  depos- 
iting a  white  sediment  at  the  bottom.  From  the  first  there  may  be  detected, 
by  reflected  light,  a  metallic  lustre  on  the  surface  of  the  colony  in  places, 
which  metallic  sheen  later  spreads  over  the  whole  colony  and  furnishes  a 
marked  differentiating  point.  In  addition  to  this,  within  twenty-four  to 
forty-eight  hours  at  37°  C.,  there  appears  a  green  fluorescence  throughout 
the  whole  of  the  medium,  which  increases  slowly  to  a  marked  bluish-green 
color,  and  never  assumes  the  nut-brown  of  the  Bacillus  pyocyaneus  of 
Gessard  upon  the  same  medium.  The  colony  is  not  especially  viscid. 

Potato. — There  appears  a  reddish-brown  colony  along  the  needle  track, 
elevated  and  moist,  confined  to  the  line  of  the  needle.  It  presents  no  change 
of  color  upon  touching  with  the  needle,  but  certain  specimens  (as  do  some  of 
the  Bacillus  pyocyaneus)  develop  later  a  heavy  green  color  extending  over 
the  whole  surface  of  the  potato,  which  later  changes  almost  to  black. 

Bouillon. — Twenty-four  hours  at  37°  C.  gives  a  growth,  especially  on  the 
surface,  which  is  a  wrinkled  scum  ;  no  cloudiness  of  the  bouillon,  and  a  very 
faint  greenish  fluorescence  one  centimetre  below  the  surface.  At  this  time 
it  differs  from  the  Bacillus  pyocyaneus  of  Gessard,  in  that  the  latter  shows 
cloudiness  of  the  medium  all  through.  Later  the  same  cloudiness  appears  in 
bouillon  cultures  of  this  new  bacillus,  together  with  a  whitish  sediment  de- 
posited at  the  bottom  of  the  tube,  and  then  the  cultures  are  indistinguishable 
from  each  other.  The  same  changes,  but  slower,  occur  at  room  tempera- 
ture. 

Peptone. — One,  3.5,  and  six-per-cent  solution.  Twenty-four  hours  at  37° 
C.  gives  a  faint  bluish  tinge  at  upper  edge  of  medium  with  very  faint  cloudi- 
ness ;  later  (in  one  or  two  weeks)  there  forms  a  marked  scum  upon  the  sur- 
face that  is  difficult  to  break  up  by  shaking,  and  the  whole  medium  assumes 
a  grass-green  color  of  more  or  less  intensity,  and  not  seen  on  other  similar 
bacilli.  The  shape  and  size  of  the  organism,  under  the  microscope,  differ 


NOT   DESCRIBED   IX   PREVIOUS   SECTIONS.  547 

very  markedly  in  this  medium  from  any  other  bacilli  examined.    The  same 
changes  are  to  be  seen  at  room  temperature,  but  more  slowly. 

Egg- Albumin:  Plain. — Twenty- four  hours  at  37°  C.,  yellowish-white, 
very  profuse  growth  all  along1  the  needle  track  ;  yellowish-green  spreading 
out  from  it  almost  to  sides  of  tube,  and  in  the  condensation  water  as  well. 
The  growth  has  no  especial  distinguishing  characteristics.  Irregular  lique- 
faction occurs,  but  the  growth  at  no  time  differs  in  any  marked  way  from 
other  varieties  of  the  Bacillus  pyocyaneus. 

Blood  Serum. — Twenty-four  hours  at  37°  C.  shows  flat,  moist  colony 
with  bluish-green  fluorescence  in  its  neighborhood.  Liquefaction  begins 
early  and  goes  on  slowly  until  complete  in  from  one  to  two  weeks,  with  an 
increasing  intensity  of  color  which  becomes  markedly  blue,  and  eventually 
almost  black. 

Milk. — Behaves  as  do  the  other  bacteria. 

BEHAVIOR  TO  TEMPERATURE. — Grows  at  15°-25°  C.  slowly;  much  more 
freely  at  35-38°  C.,  when  it  produces  the  coloi  more  quickly. 

RAPIDITY  OF  GROWTH. — Moderate. 

SPORE-PRODUCTION. — Not  observed. 

NEED  OP  AIR. — Does  not  grow  under  mica.  Facultatively  anaerobic,  but 
does  not  produce  color  except  with  free  access  of  oxygen. 

GAS-PRODUCTION. — Produces  faint  foul  odor. 

BEHAVIOR  TO  GELATIN. — Liquefies  gelatin  slowly. 

COLOR-PRODUCTION. — Produces  a  bluish-green  color  which  in  old  cul- 
tures changes  almost  to  a  black.  Upon  the  addition  of  acids  (both  vegetable 
and  mineral)  to  cultures  the  color  changes  to  red,  and  upon  the  addition  of 
alkalies  a  bright  grass-green  appears.  This  reaction  is  best  seen  in  bouillon 
and  gelatin  cultures,  but  occurs  in  other  media  as  well,  notably  blood-serum. 

BEHAVIOR  TO  ANILINE  DYES. — Stains  easily  and  well  with  any  of  the 
aniline  dyes  usually  employed,  and  by  Gram's  method. 

MICROSCOPIC  APPEARANCE  IN  DIFFERENT  MEDIA. — Under  the  micro- 
scope, its  general  appearance  on  various  media  is  of  a  rod  larger  than  the 
Bacillus  pyocyaneus.  In  peptone  cultures  this  difference  is  verv  marked. 
In  this  case,  the  Bacillus  pyocyaneus  tested  appeared  as  very  short,  oval, 
bacilli,  almost  like  micrococci,  while  the  new  bacillus  showed  as  a  long, 
fine  rod,  from  four  to  six  times  as  long  as  broad — length  about  one-half  the 
diameter  of  a  red-blood  corpuscle — and  arranged  sometimes  two  or  three 
end  to  end.  These  same  cultures  transferred  to  gelatin  became  indistin- 
guishable from  each  other  in  size. 

PATHOGENESIS. — Injections  of  small  quantities  (0.5  centimetre)  of  a  bouil- 
lon culture  twenty -four  hours  old  into  the  abdominal  cavity  of  rabbits  and 
guinea-pigs,  killed  fifty  per  cent  in  from  twenty-four  to  thirty-six  hours. 
Autopsy  showed  general  congestion  of  abdominal  viscera,  slight  effusion  into 
the  peritoneal  cavity,  and  cover-glass  preparations  and  cultures  showed  the 
bacilli  in  the  effusion  in  the  abdominal  cavity,  as  well  as  in  the  blood  from 
the  heart  and  various  organs. 

BACILLUS   OF  FIOCCA. 

Found  by  Fiocca  in  the  saliva  of  cats  and  dogs. 

Closely  resembles  the  influenza  bacillus  of  Pfeiffer  and  of  Canon. 

Morphology. — Resembles  the  bacillus  of  rabbit  septicaemia,  but  is  only 
half  as  large— from  0.2  to  0.33  /*  in  breadth.  The  length  is  but  little  greater 
than  the  breadth.  Usually  seen  in  pairs,  closely  resembling  diplococci. 
When  cultivated  on  potato  it  appears  to  be  a  micrococcus,  but  in  the  blood 
of  infected  animals  and  in  bouillon  cultures  it  is  seen  to  be  a  short  bacillus. 

Stains  with  difficulty  with  the  usual  aniline  colors,  but  is  readily  stained 
by  Ehrlich's  method  or  with  Ziehl's  solution. 

Biological  Characters.  — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  best 


548 


PATHOGENIC   AEROBIC   BACILLI 


at  37°  C.  and  does  not  develop  at  temperatures  below  15°  C.  In  agar  plates, 
at  37°  C.,  small,  punctiform  colonies  are  developed  at  the  end  of  twenty-four 
hours;  these  do  not  increase  in  size  later;  under  the  microscope  the  deep 
colonies  are  seen  to  be  spherical,  granular,  and  dark  yellow  in  color;  the 
superficial  colonies  are  more  or  less  round,  with  irregular  outlines,  trans- 
parent, slightly  granular,  and  often  have  a  shining  nucleus  at  the  centre. 
Upon  gelatin  plates  the  colonies  have  a  similar  appearance,  but  are  not  vis- 
ible in  less  than  four  or  five  days.  In  streak  cultures  upon  the  surface  of 
agar  small,  punctiform  colonies  are  seen  along  the  track  of  the  needle  at  the 
end  of  twenty-four  hours,  resembling  fine  dewdrops;  the  following  day 
these  colonies  are  a  little  larger  and  less  transparent ;  they  remain  distinct, 
especially  along  the  margins  of  the  line  of  growth.  Upon  potato  a  very 
thin,  transparent  layer  is  developed,  which  does  not  change  the  appearance 
of  the  surface  of  the  potato,  but  slightly  increases  its  resistance  to  the  plati- 
num needle.  In  bouillon  small  flocculi.  suspended  in  the  clear 'liquid,  are 
-developed  within  twenty-four  hours;  these  subsequently  sink  to  the  bottom. 

Milk  is  not  coagulated  by  this  bacillus,  and  no  gas  is  produced  in  media 
containing  sugar. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  young  rats,  and  mice, 
in  which  animals  it  produces  general  infection,  and  death— in  rabbits — at 
the  end  of  twenty-four  hours.  The  bacillus  is  found  in  the  blood  in  great 
numbers. 

PROTEUS  VULGARIS. 

Obtained  by  Hauser  (1885)  from  putrefying  animal  substances, 
and  since  shown  to  be  one  of  the  most  common  and  widely  distrib- 
uted putrefactive  bacteria.  This  and  the  other  species  of  Proteus 


Fia.  154. — Proteus  vulgaris;  "  swarming  islands  "  from  a  gelatin  culture.    X  285.    (Hauser. j 

described  by  the  same  bacteriologist  (Proteus  mirabilis,  Proteus  Zen- 
keri)  have  no  doubt  frequently  been  encountered  by  previous  observ- 
ers, and  are  among  the  species  formerly  included  under  the  name 
"  Bacterium  termo,"  which  was  applied  to  any  minute  motile  bacilli 
found  in  putrefying  infusions. 

Morphology. — Bacilli  with  rounded  ends,  about  0.6  jn  broad,  and 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  549 

varying  greatly  in  length,  being  sometimes  short  oval,  and  at  others 
from  1.25  to  3.75  /*  in  length  ;  also  grow  out  into  flexible  filaments, 
which  may  be  more  or  less  wavy  or  spiral  in  form.  The  short  rods 
are  commonly  seen  in  pairs  ;  they  have  terminal  flagella  ;  involution 
forms  are  frequently  seen,  the  most  common  being  spherical  bodies 
about  1.6  jit  in  diameter.  In  old  cultures  in  bouillon,  or  in  cultures 
made  in  meat  infusion  in  the  incubating  oven,  the  short  oval  forms 
greatly  predominate,  but  in  recent  cultures  in  nutrient  gelatin  fila- 
ments of  considerable  length  are  encountered  in  association  with 
shorter  rods. 

Stains  readily  with  fuchsin  or  gentian  violet — not  so  well  with 
the  brown  aniline  colors  ;  does  not  stain  by  Gram's  method  (Cheyne). 

Biological  Characters. — Anaerobic  and  facultative  anaerobic, 
liquefying,  motile  bacillus.  Grows  rapidly  in  the  usual  culture 
media  at  the  room  temperature. 

The  growth  upon  gelalin  plates  (five  per  cent  of  gelatin)  at  the 
room  temperature  is  very  characteristic  ;  at  the  end  of  six  or  eight 
hours  small  depressions  in  the  gelatin  are  observed,  which  contain 
liquefied  gelatin  and  grayish- white  masses  of  bacilli.  Under  a  low 
power  these  depressions  are  seen  to  be  surrounded  by  a  marginal 
zone  consisting  of  two  or  three  layers,  outside  of  which  is  a  zone  of  a 
single  layer,  from  which  amoeba-like  processes  extend  upon  the  sur- 
face of  the  gelatin.  These  processes  are  constantly  undergoing 
changes  in  their  form  and  position,  and  may  become  separated  from 
the  mother  colony,  or  remain  temporarily  attached  to  it  by  a  narrow 
thread  consisting  of  bacilli ;  after  a  time  the  entire  surface  of  the 
gelatin  is  covered  with  wandering,  amoeba-like  colonies ;  these 
rapidly  cause  liquefaction,  which  by  the  end  of  twenty-four  to  forty- 
eight  hours  has  reached  a  depth  of  one  millimetre  or  more  over  the 
entire  surface.  The  deep  colonies  also  are  surrounded  by  processes 
projecting  into  the  gelatin,  which  may  be  observed  to  suddenly  ad- 
vance and  again  to  be  retracted  towards  the  central  zoogloea-like 
mass.  Liquefaction  around  the  colony  rapidly  progresses,  and 
actively  motile  rods  and  spiral  filaments  may  be  seen  about  the  peri- 
phery of  this  liquefied  gelatin,  while  about  it  is  a  radiating  crown  of 
irregular  processes,  some  of  which  may  be  screw-like  or  corkscrew- 
formed.  In  ten-per-cent  gelatin  the  migration  of  surface  colonies, 
above  described,  is  not  observed.  In  gelatin  stab  cultures  liquefac- 
tion occurs  along  the  entire  line  of  puncture,  and  soon  the  contents 
of  the  tube  are  completely  liquefied  ;  near  the  surface  of  the  liquefied 
gelatin  the  growing  bacilli  form  a  grayish-white  cloudiness,  and  at 
the  bottom  of  the  tube  an  abundant  flocculent  deposit  is  formed. 
Upon  the  surface  of  nutrient  agar  a  rapidly  extending,  moist,  thin, 
grayish- white  layer  is  formed.  Upon  potato  this  bacillus  produces  a 


550  PATHOGENIC   AEROBIC   BACILLI 

dirty -white,  moist  layer.  The  cultures  in  media  containing  albumin 
or  gelatin  have  a  putrefactive  odor  and  acquire  a  strongly  alkaline 
reaction.  A  temperature  of  20°  to  24°  C.  is  most  favorable  for  the 
growth  of  this  bacillus.  It  is  a  facultative  anaerobic  and  grows  in 
an  atmosphere  of  hydrogen  or  of  carbon  dioxide,  although  not  so 
rapidly  as  in  the  presence  of  oxygen.  The  movements  are  often  ex- 
tremely active  and  difficult  to  follow  under  the  microscope  ;  again 
they  may  be  quite  deliberate,  or  the  bacilli  may  remain  motionless 
for  a  time  and  again  dart  off  in  active  motion.  The  long  terminal 
flagella  may  sometimes  be  discerned  by  means  of  a  good  objective 
and  careful  manipulation  of  the  light. 

Pathogenesis. — Pathogenic  for  rabbits  and  for  guinea-pigs  when 
injected  into  the  circulation,  into  the  cavity  of  the  abdomen,  or  sub- 
cutaneously  in  considerable  quantity.  Cultures  in  nutrient  gelatin 
are  said  by  Cheyne  to  be  more  pathogenic  (toxic)  than  those  in  bouil- 
lon. When  injected  into  the  muscles  of  rabbits  a  much  smaller 
dose  produces  a  fatal  result  than  when  injected  subcutaneously. 
In  Cheyne's  experiments,  made  in  London  (1886),  one-tenth  cubic 
centimetre  of  a  liquefied  gelatin  culture,  injected  into  the  dorsal 
muscles,  was  invariably  fatal  in  from  twenty-four  to  thirty-six  hours; 
a  dose  of  one-twentieth  cubic  centimetre,  injected  in  the  same  way, 
usually  caused  death;  while  one-fortieth  cubic  centimetre  gave  rise  to 
an  extensive  local  abscess,  and  the  animals  died  at  the  end  of  six  or 
eight  weeks.  Doses  of  less  than  one-five-hundredth  cubic  centimetre 
produced  no  effect.  Cheyne  estimates  that  one  cubic  centimetre  of  a 
culture  in  nutrient  gelatin  contains  4,500,000,000  bacilli,  and,  conse- 
quently, that  a  smaller  number  than  9,000,000  produced  no  effect  when 
injected  into  the  muscular  tissue  of  rabbits.  Injections  into  the  sub- 
cutaneous connective  tissues  of  a  dose  twice  as  large  as  that  which  in- 
variably proved  fatal  when  injected  into  the  muscles  usually  caused 
an  extensive  abscess,  but  did  not  kill  the  animal ;  and,  after  re- 
covery from  the  effects  of  such  an  injection,  the  rabbit  was  found  to 
be  immune  against  a  similar  dose  injected  into  the  muscles.  Foa 
and  Bonome  have  succeeded  in  producing  immunity  against  the 
effects  of  virulent  cultures  of  this  bacillus  by  inoculating  rabbits  with 
filtered  cultures,  and  also  by  injecting  beneath  the  skin  of  these  ani- 
mals a  solution  of  neurin,  which  they  believe  to  be  the  principal 
toxic  product  present  in  the  cultures. 

Proteus  Vulgar  is  in  Cholera  Infantum. — The  extended  re- 
searches of  Booker  have  led  him  to  the  conclusion  that  this  bacillus 
plays  an  important  part  in  the  production  of  the  morbid  symptoms 
which  characterize  cholera  infantum.  Proteus  vulgaris  was  found 
in  the  alvine  discharges  in  a  considerable  proportion  of  the  cases  ex- 
amined, but  was  not  found  in  the  faeces  of  healthy  infants.  '  The 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  551 

prominent  symptoms  in  the  cases  of  cholera  infantum  in  which  the 
proteus  bacteria  were  found  were  drowsiness,  stupor,  emaciation 
and  great  reduction  in  flesh,  more  or  less  collapse,  frequent  vomiting 
and  purging,  with  watery  and  generally  offensive  stools." 

The  researches  of  Krogius,  Schnitzler,  Schmidt  and  Aschoff,  and 
others,  show  that  in  cases  of  cystitis  and  of  pyelonephritis  this  bacil- 
lus is  frequently  found  in  pure  cultures,  or  associated  with  other  bac- 
teria. The  authors  last  named  state  that  in  sixty  cases  of  cystitis 
reported  by  various  authors  the  colon  bacillus  was  found  in  pure  cul- 
tures, and  in  thirteen  cases  the  proteus  of  Hauser.  Next  to  Bacillus 
coli  communis  Proteus  vulgaris  appears  to  be  the  microorganism 
most  frequently  concerned  in  the  etiology  of  pyelonephritis. 

Levy  (1895)  isolated  from  sour  yeast  a  bacillus,  which  he  identified 
as  "Proteus  Hauseri,"  and  made  numerous  experiments  on  dogs  to 
test  its  pathogenic  power.  From  five  to  ten  cubic  centimetres  of  a 
liquefied  gelatin  culture  injected  into  the  circulation,  through  a  vein, 
caused  the  typical  symptoms  of  "sepsin  poisoning,"  as  formerly  de- 
scribed by  Bergmann  and  Schmeideberg  (1868).  In  two  dogs  which 
died  at  the  end  of  forty-eight  hours  the  intestinal  tract  was  found  in 
a  condition  of  intense  hemorrhagic  infiltration.  The  spleen  and 
glands  of  the  mesentery  were  much  enlarged.  But  a  bacteriological 
examination  gave  an  entirely  negative  result,  showing  that  death 
resulted  from  toxemia  and  not  from  septica3mia.  Further  experi- 
ments showed  that  the  dried  precipitate  obtained  from  liquefied  gela- 
tin cultures,  by  the  addition  of  alcohol,  had  the  same  pathogenic 
action  on  dogs,  rabbits,  and  mice  as  cultures  containing  the  living 
bacilli.  That  a  similar  pathogenic  effect  is  produced  in  man  by  the 
products  of  growth  of  this  bacillus  was  shown  by  the  following  facts : 
While  conducting  his  experiments  Levy  had  an  opportunity  to  make 
a  bacteriological  examination  in  the  case  of  a  man  who  died  after  a 
brief  attack  of  cholera  morbus.  From  the  vomited  material  and  the 
stools  he  obtained  a  pure  culture  of  proteus ;  but  the  blood,  collected 
at  the  autopsy,  was  sterile.  In  the  mean  time  seventeen  other  per- 
sons who  had  eaten  at  the  same  restaurant  were  taken  sick  in  the  same 
way.  Upon  an  examination  at  the  restaurant  it  was  found  that  the 
bottom  of  the  ice  chest  in  which  the  proprietor  kept  his  meats  was 
covered  with  a  slimy,  brown  layer,  which  gave  off  a  disagreeable 
odor.  Cultures  from  this  gave  the  proteus  as  the  principal  micro- 
organism present.  Levy  concludes  from  his  own  investigations  and 
those  of  other  bacteriologists  that  in  so-called  "  flesh-poisoning  "  bac- 
teria of  this  group  are  chiefly  at  fault,  and  that  the  pathogenic  effects 
are  due  to  toxic  products  evolved  during  their  development. 


PATHOGENIC   AEROBIC   BACILLI 

PROTEUS  OF   KARLINSKI. 

Synonym.—  Bacillus  murisepticus  pleomorphus  (Karlinski).  Probe  bly 
identical  with  Proteus  vulgaris  of  Hauser. 

Obtained  by  Karlinski  (1889)  from  a  fibro-purulent  uterine  discharge,  and 
from  abscesses  in  the  uterus  and  its  appendages  in  a  puerperal  woman. 

Morphology.  — Eesembles  Proteus  vulgaris  of  Hauser  in  its  morphology, 
and  presents  various  forms  under  different  circumstances  relating  to  the 
culture  medium,  the  temperature,  age  of  culture,  etc. — sometimes  as  spheri- 
cal or  short  oval  cells,  at  others  as  longer  or  shorter  rods  or  spiral  filaments; 
usually  as  bacilli  with  round  ends  two  and  a  half  times  as  long  as  thick,' 
often  united  in  pairs. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Spore  formation  not  observed.  Grows  rapidly  in  the 
usual  culture  media  at  the  room  temperature.  In  gelatin  plate  cultures,  at 
the  end  of  ten  hours,  small  colonies  are  developed  which  have  well-defined 
outlines,  are  oval  or  whetstone-shaped,  of  a  light-brown  color  by  transmitted 
light  and  white  by  reflected  light,  with  a  somewhat  darker  margin  and  a 
smooth  surface,  sometimes  marked  by  shallow  clefts ;  at  the  end  of  twenty 
hours  the  colonies  commence  to  have  irregular  margins,  and  the  surface  of 
the  gelatin  above  them  is  marked  by  concentric  rings.  At  the  end  of  thirty 
hours  the  colonies  have  formed  a  bulb-shaped  liquefaction  of  the  gelatin, 
and  delicate,  ray-like  offshoots  are  seen  around  the  margin.  At  the  end  of 
two  days  the  bulbous  cavities  are  aboutone  and  a  half  millimetres  in  diameter 
and  contain  a  cloudy,  grayish-white  liquid;  they  are  surrounded  by  a  moist- 
looking,  gray,  irregular  marginal  zone.  In  gelatin  stab  cultures,  at  the  end 
of  twenty-four  hours,  a  funnel-shaped  liquefaction  of  the  gelatin  occurs  near 
the  surface,  and  a  grayish-white,  cloudy  mass  is  developed  along  the  line  of 
puncture;  at  the  end  of  forty- eight  hours  a  sac-like  pouch  of  liquefied  gela- 
tin has  formed,  and  in  the  course  of  four  or  five  days  the  gelatin  is  entirely 
liquefied.  Upon  agar  plates  the  colonies  are  at  first  oval  in  form  and  white 
by  reflected  light,  or  pale  brown  by  transmitted  light  ;  at  the  end  of  thirty 
hours  the  surface  becomes  wrinkled  or  folded  and  is  surrounded  by  radiat- 
ing, delicately  twisted  offshoots.  Upon  the  surface  of  agar  a  white  layer 
is  developed.  Upon  potato  a  whitish-gray,  soft,  homogeneous  layer,  which 
after  standing  along  time  has  a  darker  color.  Upon  blood  serum  a  thin, 
grayish-white  layer  is  formed  and  the  serum  is  rapidly  liquefied.  Gelatin 
cultures  acquire  a  strongly  alkaline  reaction  and  give  off  a  disagreeable 
odor  resembling  that  of  butyric  acid. 

Pathogenesis. — White  mice  inoculated  at  the  root  of  the  tail  die  in  from 
twenty-two  to  twenty-four  hours  ;^the  spleen  is  greatly  enlarged;  the  bacilli 
are  found  in  blood  from  the  various  organs — less  numerous  in  blood  from 
the  heart.  Field  mice  and  house  mice  are  less  susceptible.  Subcutaneous 
injections  in  rabbits  may  give  rise  to  local  inflammation  and  also  to  general 
infection.  In  white  rats  and  guinea-pigs  a  local  abscess  may  result  from  a 
subcutaneous  inoculation. 

PROTEUS   MIRABILIS. 

Obtained  by  Hauser  (1885)  from  putrefying  animal  substances. 

Morphology. — Bacilli  resembling  very  closely  the  preceding  species  (Pro- 
teus vulgaris),  but  presenting  more  numerous  involution  forms,  which  may' 
be  spherical,  pear-shaped,  or  spermatozoa-like,  etc.  The  bacilli  are  about 
0.6  n  in  diameter  and  vary  greatly^in  length,  being  sometimes  nearly  spheri- 
cal, or  forming  rods  of  2  to  3.75  /*  in  length,  or  long  filaments. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Spore  formation  has  not  been  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Does  not  liquefy  gelatin  as 


NOT   DESCRIBED    IN   PREVIOUS   SECTIONS. 


553 


rapidly  as  Proteus  vulgaris.  Upon  gelatin  plates,  at  the  end  of  twelve 
hours,  superficial  colonies  of  two  to  three  millimetres  in  diameter  are  formed ; 
under  a  low  power  these  appear  finely  granular  and  brownish  in  color,  and 
have  an  irregular  outline;  outgrowths  from  the  margin  extend  in  various 
directions  and  form  new  colonies,  which  may  be  attached  for  a  time  by  a 
long  and  slender  thread  consisting  of  bacilli.  The  movement  of  these  new 
colonies  is  not  as  pronounced  as  in  the  case  of  the  preceding  species,  and 


FIG.  155. — "  Swarming  islands  "  of  Proteus  mirabilis,  from  a  gelatin  culture,    x  285.    (Hauser.) 

they  are  characterized  by  the  presence  of  numerous  distorted  bacilli — invo- 
lution forms.     The  deep  colonies  form  spiral  zooglcea  masses. 

In  gelatin  stab  cultures  the  whole  surface  is  first  covered  with  threads 
and  islands  of  bacilli,  which  after  a  time  form  an  anastomosing  network,  and 
finally  a  confluent  layer  which  at  the  end  of  forty-eight  hours  is  rather  thick, 


"FiG.  156.— Spiral  zooglcea  from  a  culture  of  Proteus  mirabilis.     X  95.    (.Hauser.) 

with  a  moist,  shining  surface  and  grayish  color,  and  appears  to  be  perforated 
with  numerous  small,  sieve-like  openings.  These  thinner  and  transparent 
places  disappear  after  a  time,  and  at  the  end  of  two  or  three  days  liquefac- 
tion of  the  gelatin  commences;  complete  liquefaction  does  not  occur  until 
the  fifth  or  sixth  day,  or  even  later.  Along  the  line  of  puncture  finely  gran- 
ular colonies  are  first  formed,  from  which  long  threads  are  given  off,  which 
form  after  a  short  time  a  tolerably  broad  zone  of  threads  and  spiral  zooglcea 
masses. 


554  PATHOGENIC   AEROBIC   BACILLI 

Pathogenesis. — In  Hauser's  experiments  filtered  cultures  (two  to  six  cubic 
centimetres),  injected  into  the  circulation  or  into  the  cavity  of  the  abdomen 
in  rabbits,  caused  fatal  toxaemia. 

PROTEUS  ZENKERI. 

Obtained  by  Hauser  (1885)  from  putrefying  animal  substances. 

Morphology. — Bacilli  which  vary  greatly  in  length — average  about  1. 65  /*, 
and  about  0.4  ju  broad. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  motile  bacillus.  Spore  formation  not  observed.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Upon  the  surface  of  nutrient 
gelatin  a  laminated  mass  forms  about  the  point  of  puncture,  from  the  peri- 
phery of  which  offshoots  are  given  off,  at  the  extremities  of  which  colonies 
are  formed,  as  in  the  case  of  Proteus  mirabilis.  Gradually  a  rather  thick, 
grayish-white,  opaque  layer  is  formed,  which  covers  the  entire  surface  of  the 
gelatin  and  is  easily  detached  from  it.  This  species  is  distinguished  from 
the  two  preceding  by  the  fact  that  it  does  not  liquefy  gelatin  or  blood  serum 
and  does  not  give  off  a  decided  putrefactive  odor  when  cultivated  in  these 
^nedia. 

Pathogenesis. — Considerable  quantities  injected  into  small  animals  give 
rise  to  local  abscesses  and  to  symptoms  of  toxaemia. 

PROTEUS   SEPTICUS. 

Obtained  by  Babes  (1889)  from  the  mucous  membrane  of  the  intestine  and 
the  various  organs  of  a  boy  who  died  of  septicaemia. 

Morphology. — Bacilli  about  0. 4  ju.  broad  and  varying  greatly  in  length; 
slightly  curved  rods  or  flexible  filaments,  often  associated  in  loose  chains. 

Stains  by  the  usual  aniline  colors  and  by  Gram's  method. 

Biological  Characters. — An  aerobic,  liquefying,  motile  bacillus.  Spore 
formation  not  observed.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  In  gelatin  plates  centres  of  liquefaction  are  quickly  formed 
and  rapidly  extend.  The  spherical,  liquefied  places  have  at  first  a  wavy  or 
dentate  outline,  and  are  surrounded  by  a  branching,  transparent,  granular 
margin  which  rapidly  extends  in  advance  of  the  liquefaction.  In  stab  cul- 
tures in  nutrient  gelatin  liquefaction  of  the  entire  contents  of  the  tube  may 
take  place  within  twenty-four  hours,  or  a  broad,  liquefied  sac  is  formed 
along  the  line  of  puncture.  Gelatin  cultures  give  off  a  very  disagreeable 
•odor.  Upon  the  surface  of  nutrient  agar,  at  37°  C.,  a  peculiar,  thick  net- 
work extends  over  the  surface  in  the  course  of  a  few  hours.  Upon  potato  an 
^elevated,  brownish-white,  shining  layer  is  formed.  Blood  serum  is  lique- 
fied by  this  bacillus. 

Pathogenesis. — Pathogenic  for  mice,  less  so  for  rabbits.  In  mice  death 
occurs  in  from  one  to  three  days  after  the  subcutaneous  injection  of  a  small 
quantity  of  a  pure  culture  ;  the  bacilli  are  present  in  the  blood  in  small 
numbers. 

PROTEUS   LETHALIS. 

Synonym. — Proteus  bei  Lungengangran  des  Menschen  (Babes). 

Obtained  by  Babes  (1889)  from  the  spleen  and  gangrenous  portions  of  the' 
lung  of  a  man  who  died  of  septicaemia. 

Morphology. — Short  rods  with  round  ends,  from  0.8  to  1.5  ju  thick  ;  often 
•swollen  in  the  middle,  like  a  lemon  or  a  flask  ;  forms  short,  flexible  filaments 
which  also  present  similar  swellings. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
fying,  motile  bacillus.  Not  observed  to  form  spores.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  In  gelatin  plates  forms  hemi- 
spherical, elevated,  whitish,  translucent  colonies,  which  later  send  out 


NOT  DESCRIBED  IN  PREVIOUS   SECTIONS.  555 

coarse  branches  which  ramify  over  the  surface  of  the  gelatin.  •  A  similar 
growth  is  observed  upon  the  surface  of  gelatin  stab  cultures,  and  an  abun- 
dant development  takes  place  along  the  line  of  puncture.  Upon  nutrient 
agar  a  thick,  opaque,  slightly  yellowish  layer  is  formed.  Upon  potato  a 
moist,  shining,  brownish  layer  is  developed,  and  the  potato  acquires  a 
brownish  color.  Upon  blood  serum  the  growth  is  less  abundant  than  on 
agar;  the  blood  serum  is  not  liquefied.  This  bacillus  grows  rapidly  at  the 
room  temperature;  it  is  destroyed  by  a  temperature  of  80°  C.,  and  presum- 
ably does  not  form  spores. 

Pathogenesis. — Recent  cultures  are  very  pathogenic  for  mice  and  for 
rabbits,  less  so  for  guinea-pigs.  The  subcutaneous  injection  of  a  small 
quantity  of  a  pure  culture  kills  susceptible  animals  in  two  or  three  days. 
More  or  less  oedema  is  found  at  the  point  of  inoculation.  Injections  into  the 
rectum  of  rabbits  gave  rise  to  haemorrhagic  enteritis,  peritonitis,  and  death 
at  the  end  of  four  days. 

BACILLUS   A   OF  BOOKER. 

Obtained  by  Booker  (1889)  from  the  alvine  discharges  of  children  suffer- 
ing from  cholera  infantum. 

Morphology. — Bacilli  with  round  ends,  varying  greatly  in  length,  usually 
three  to  four  //  long  and  0.7  /*  broad  (in  recent  agar  cultures).  In  older  cul- 
tures the  bacilli  are  shorter  and  smaller. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  lique- 
fying, motile  bacillus.  Grows  at  the  room  temperature  in  the  usual  culture 
media.  In  gelatin  plates  colonies  are  visible  at  the  end  of  twenty -four 
hours;  under  the  microscope  these  are  nearly  colorless,  and  liquefaction 
•soon  occurs  around  them.  In  gelatin  stab  cultures  complete  liquefaction 
occurs  in  three  or  four  days .  Upon  agar  a  colorless  layer  covering  the  entire 
surface  is  developed  in  three  or  four  days,  and  an  abundant  development 
occurs  along  the  line  of  puncture.  Agar  colonies  have  a  bluish  look,  and 
are  surrounded  by  an  indistinct  halo  which  shades  off  gradually  into  the 
surrounding  agar  ;  under  a  low  power  the  colonies  are  light-brown  and  the 
borders  indistinct ;  the  surface  has  a  delicate,  wavy  appearance.  Upon  po- 
tato the  growth  is  luxuriant  and  of  a  dirty-brown  color.  Blood  serum  is 
liquefied  by  this  bacillus. 

Milk  is  coagulated  into  a  gelatinous  mass  having  an  alkaline  reaction ; 
later  the  coagulum  is  dissolved. 

Pathogenesis. — Mice  and  guinea-pigs  fed  with  cultures  in  milk  die  in  from 
one  to  eight  days. 

BACILLUS   ENDOCARDITIDIS   GRISEUS. 

Obtained  by  Weichselbaum  (1888)  from  the  affected  valves  in  a  case  of 
endocarditis  recurrens  ulcerosa. 

Morphology. — Short  rods  with  rounded  or  somewhat  pointed  ends,  about 
two  to  three  times  as  long  as  broad — of  about  the  same  dimensions  as  the 
bacillus  of  typhoid  fever. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method;  the 
longer  rods  from  old  cultures  are  irregularly  stained. 

Biological  Characters. — An  aerobic,  non-liquefying,  motile  bacillus. 
Refractive  bodies  may  be  seen  in  some  of  the  rods,  which  resemble  spores  and 
are  stained  by  the  method  of  Ernst,  but  they  do  not  show  the  resistance  of 
known  spores  to  physical  and  chemical  agents.  Grows  well  in  the  usual 
culture  media  at  the  room  temperature.  Upon  gelatin  plates  colonies  are 
formed  which  resemble  those  of  Friedlander's  bacillus,  but  which  gradually 
acquire  a  gray  or  grayish- white  color.  The  prominent,  convex,  superficial 
colonies  under  a  low  power  are  finely  granular  and  grayish -brown  in  color; 
the  deep  colonies  are  yellowish-brown  in  color,  have  slightly  notched  mar- 
gins, and  the  surface  is  covered  with  minute  projections.  In  stab  cultures 


PATHOGENIC   AEROBIC   BACILLI. 

a  rather  thin,  circular  layer  forms  about  the  point  of  puncture ;  this  has  the 
appearance  of  stearin ;  later  it  becomes  grayish- white  and  the  margins  are 
marked  by  radiating  lines.  Upon  the  surface  of  nutrient  agar  a  similar 
growth  occurs  which  has  a  pale-brown  or  reddish-gray  color.  Upon  potato 
in  the  incubating  oven  an  abundant  development  occurs,  forming  a  dry- 
looking  layer  of  a  grayish-brown  color  and  having  irregularly  notched  mar- 
gins. Upon  blood  serum  an  abundant,  grayish-white  growth  of  cream-like 
consistence  forms  along  the  impfstrich ;  later  this  has  a  reddish  gray  color. 
This  bacillus  grows  to  the  bottom  of  the  line  of  puncture  in  stick  cultures, 
and  is  no  doubt  a  facultative  anaerobic. 

Pathogenesis. — Pathogenic  for  white  mice  and  for  guinea-pigs. 

BACILLUS   ENDOCARDITIDIS   CAPSULATUS. 

Obtained  by  Weichselbaum  (1888)  from  thrombi  and  embolic  infarctions 
in  the  spleen  and  kidneys  of  a  man  who  died  from  endocarditis  with  forma- 
tion of  thrombi. 

Morphology. — Resembles  Friedlander's  bacillus,  and  is  frequently  sur- 
rounded by  a  capsule,  which  may  be  stained ;  also  forms  long,  curved  fila- 
ments, in  the  protoplasm  of  which  vacuoles  may  be  observed  in  stained  pre- 
parations. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method ;  by 
staining  with  fuchsin  arid  carefully  decolorizing  with  diluted  alcohol  the 
presence  of  a  capsule  may  be  demonstrated. 

Biological  Characters.— An  aerobic,  no n-liquefying  bacillus.  Grows  in 
the  usual  culture  media  at  the  room  temperature. 

In  gelatin  stab  cultures  development  occurs  along  the  line  of  puncture, 
and  on  the  surface  as  a  rather  thin,  white,  dry  layer  which  resembles  stearin. 
In  agar  plates  the  superficial  colonies  are  thin,  about  two  millimetres  in 
diameter  and  gray  in  color  ;  under  a  low  power  the  margins  are  trans- 
parent and  colorless,  and  the  centre  resembles  the  deep  colonies;  these  are 
very  small  and  grayish- white  in  color  ;  under  a  low  power  the  surface  is 
seen  to  be  covered  with  tooth-like,  projecting  masses,  the  margin  is  dentate 
and  has  a  pale-yellow  color,  while  the  centre  is  yellowish- brown. 

Pathogenesis. — Rabbits  are  killed  by  the  injection  of  a  considerable  quan- 
tity of  a  pure  culture  into  the  cavity  of  the  abdomen  or  subcutaneously. 

BACILLUS   ALVEI. 

Synonym. — Bacillus  of  foul  brood  (of  bees). 

Obtained  by  Cheshire  and  Cheyne  (1885)  from  the  larvae  in  hives  infected 
with  "  foul  brood."  The  larvae  in  the  interior  of  cells  in  the  comb  die  and 
become  almost  fluid  as  a  result  of  parasitic  invasion  by  this  bacillus. 

Morphology.—  Bacilli  with  rounded  ends,  from  2.5  to  5  ju  in  length  (aver- 
age about  3.6  //)  and  0.8  u  in  diameter.  Grow  out  into  filaments  and  form, 
large  oval  spores  which  have  a  greater  diameter  than  the  rods  in  which  they 
are  developed — 1.07  n. 

Stains  readily  with  the  aniline  colors  usually  employed,  also  by  Gram's 
method. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  bacillus.  Forms  endogenous  spores.  Grows  readily  in  the  usual 
culture  media  at  the  room  temperature. 

In  gelatin  plates  small,  round  or  oval  colonies  are  formed,  which  later 
become  pear-shaped;  a  branching  outgrowth  occurs  about  the  margins  of  the 
colonies,  and  especially  from  the  small  end  of  the  pear-shaped  mass.  In 
streak  cultures  upon  the  surf  ace  of  gelatin  growth  occurs  first  along  the  impf- 
strich, and  from  this  an  outgrowth  occurs  consisting  of  bacilli  in  a  single 
row  or  in  several  parallel  rows,  and  forming  irregular  or  circular  figures 


NOT   DESCRIBED   IN  PREVIOUS   SECTIONS.  557 

from  which  other  similar  outgrowths  occur  ;  the  branching1  outgrowths  may 
anastomose.  The  gelatin  is  liquefied  in  the  vicinity  of  these  lines  of  growth, 
forming  a  network  of  channels.  A  similar  growth  is  seen  upon  the  surface 
of  gelatin  stab  cultures,  and  along  the  line  of  puncture  white,  irregular 
masses  are  formed,  from  which  rather  coarse  branches  are  given  off  which 
often  have  a  club-shaped  extremity.  In  older  cultures  the  finer  branches 
disappear,  so  that  the  secondary  centres  of  growth  are  disconnected  from  the 
original  colonies ;  complete  liquefaction  of  the  gelatin  occurs  in  about  two 
weeks;  the  liquefied  gelatin  has  a  yellowish  color  and  peculiar  odor.  Upon 
the  surface  of  nutrient  agar,  at  37°  C.,  a  white  layer  is  formed.  Upon  potato 
the  development  is  slow  and  results  in  the  formation  of  a  dry,  yellowish 
layer.  In  milk  coagulation  first  occurs,  and  the  coagulum  is  subsequently 
dissolved;  a  slightly  acid  reaction  is  produced.  This  bacillus  grows  best  in 
the  incubating  oven,  at  37°,  and  does  not  develop  at  temperatures  below  16° 
C.  The  spores  require  for  their  destruction  a  temperature  of  100°  C.  main- 
tained for  four  minutes  (determined  by  the  writer,  1887). 

Pathogenesis. — The  introduction  of  pure  cultures  of  this  bacillus  into 
hives  occupied  by  healthy  swarms  causes  them  to  become  infected  with  foul 
brood;  grown  bees  also  become  infected  when  given  food  containing  the  ba- 
cillus (Cheshire) .  Mice  injected  subcutaiieously  with  a  considerable  quan- 
tity die  within  twenty-four  hours,  guinea-pigs  in.  six  days  (Eisenberg). 
Small  amounts  injected  beneath  the  skin  of  mice  or  rabbits  produce  no  appa- 
rent result. 

BACILLUS   OP   ACNE   CONTAGIOSA  OF   HORSES. 

Obtained  by  Dieckerhoff  and  Grawitz  (1885)  from  pus  and  dried  scales 
from  the  pustules  of  "  acne  contagiosa  "  of  horses. 

Morphology.— Short  rods,  straight  or  slightly  bent,  0.2  /*  in  diameter. 

Stains  best  with  an  aqueous  solution  of  fuchsin,  and  also  by  Gram's 
method ;  does  not  stain  well  with  Loffler's  alkaline  solution  of  methylene 
blue. 

Biological  Characters. — Anaerobic,  non-liquefying  bacillus.  In  gelatin 
stab  cultures  a  very  scanty  growth  occurs  along  the  line  of  puncture ;  upon 
the  surface  a  white  mass  forms  about  the  point  of  puncture.  Upon  blood 
serum  and  nutrient  agar  an  abundant  growth  at  the  end  of  twenty -four 
hours  at  37°  C.,  consisting  of  white  colonies  along  the  impfstrich,  which 
later  have  a  yellowish-gray  color.  The  growth  is  more  abundant  and  rapid 
upon  blood  serum  than  upon  other  media. 

Pathogenesis. — Pure  cultures  of  the  bacillus  described  are  said  by  Diecker- 
Tioff  and  Grawitz  to  produce  typical  acne  pustules  when  rubbed  into  the  skin 
of  horses,  calves,  sheep,  and  dogs.  When  rubbed  into  the  intact  skin  of 
guinea-pigs  a  phlegmonous  erysipelatous  inflammation  was  produced,  and 
the  animal  died  at  the  end  of  forty-eight  hours  with  symptoms  of  toxaemia. 
Subcutaneous  injections  in  guinea-pigs  caused  toxaemia  and  death  at  the  end 
of  twenty-four  hours.  At  the  autopsy  a  haemorrhagic  infiltration  of  the  in- 
testinal mucous  membrane  was  observed ;  the  bacilli  were  not  found  in  the 
internal  organs.  In  rabbits  pure  cultures  rubbed  into  the  intact  skin  caused 
a  development  of  pustules  and  a  severe  inflammation  of  the  subcutaneous 
connective  tissue,  from  which  the  animal  usually  recovered.  Subcutaneous 
injections  in  rabbits  sometimes  caused  a  fatal  toxaemia.  House  mice,  field 
mice,  and  white  mice  were  not  affected  by  the  application  of  cultures,  by 
rubbing,  to  the  uninjured  skin,  but  succumbed  to  subcutaneous  injections  in 
twenty-four  hours  or  between  the  fifth  and  tenth  days.  Those  which  died 
at  a  late  date  presented  the  pathological  appearances  which  characterize 
pyaemia. 


558  PATHOGENIC   AEROBIC   BACILLI 


BACILLUS  OF  PURPURA  HJEMORRHAGICA   OF   TIZZONI  AND   GIO- 

VANNINI. 

Obtained  by  Tizzoni  and  Giovannini  (1889)  from  the  blood  of  two  children, 
who  died  of  purpura  haemorrhagica  following  impetigo. 

Morphology. — Bacilli  with  round  ends,  from  0.75  to  1.3  /*  long  and  0.2 
to  0.4  yu  broad;  often  seen  in  pairs  or  in  groups  like  streptococci. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Spore  formation  not  observed.  Grows  in 
the  usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  the 
colonies  at  first  resemble  those  of  Streptococcus  pyogenes.  Upon  the  surface 
small,  opaque  points  are  seen  at  the  end  of  >  forty-eight  hours,  which  at  the 
end  of  four  to  five  days  develop  into  spherical,  yellowish-gray  colonies  with 
irregular  margins,  surrounded  by  a  growth  resembling  tufts  of  curly  hair. 
Upon  agar  the  growth  is  similar,  but  more  rapid  and  of  a  pale  color,  often 
with  a  central  nucleus  surrounded  by  a  net-like  marginal  zone.  Upon 
blood  serum  the  growth  is  similar  to  that  upon  agar.  Upon  potato,  at  37° 
C.,  a  limited  development  occurs  about  the  point  of  inoculation,  which  has 
a  dark-yellow  color.  The  cultures  give  off  a  very  penetrating  odor. 

Pathogenesis. — Pathogenic  for  dogs,  rabbits,  and  guinea-pigs  when  in- 
jected subcutaiieously.  Not  pathogenic  for  white  mice  or  pigeons.  The 
symptoms  resulting  from  a  subcutaneous  injection  are  said  to  be  fever,  al- 
buminuria  and,  in  some  cases,  anuria,  haemorrhagic  spots  upon  the  skin, 
convulsions  ;  death  occurs  in  from  one  to  three  days.  At  the  autopsy  there 
are  found  oedema  about  the  point  of  inoculation ,  haemorrhages  in  the  skin  and 
muscles,  and  sometimes  in  the  internal  organs  and  in  serous  cavities;  the 
blood  does  not  coagulate.  The  bacilli  are  found  in  the  subcutaneous  con- 
nective tissue,  but  not  in  the  blood  or  in  the  various  organs.  Sections  show 
coagulation  necrosis  of  the  liver  cells  and  of  the  renal  epithelium. 

BACILLUS   OF  PURPURA   H^EMORRHAGICA  OF  BABES. 

Obtained  by  Babes  (1890)  from  the  spleen  and  lungs  of  an  individual  who 
died  from  purpura  haemorrhagica  with  symptoms  of  septicaemia.  Resembles 
the  bacillus  previously  described  by  Tizzoni  and  Giovannini,  and  still  more 
that  of  Kolb ;  but,  according  to  Babes,  differs  in  some  respects  from  both  of 
these,  although  they  all  belong  evidently  to  the  same  group. 

Morphology. — Bacilli  with  rounded  ends,  oval  or  pear-shaped,  about  0.3  /* 
thick,  surrounded  by  a  narrow  capsule. 

Stains  with  the  aniline  colors,  but  not  deeply,  and  still  less  intensely  by 
Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  In  gelatin  stick  cultures,  at  the 
end  of  three  days,  a  thin,  transparent,  irregular  layer  has  developed  upon 
the  surface,  and  a  whitish,  punctate  stripe  along  the  line  of  inoculation.  In 
agar  stick  cultures  an  abundant  development  occurs  along  the  line  of  punc- 
ture, and  at  the  end  of  three  days  the  growth  upon  the  surface  consists  of 
small,  moist,  transparent  drops;  later  of  larger,  flat,  shining,  yellowish- 
white  plaques  which  have  ill-defined  margins.  Upon  blood  serum  the  de- 
velopment is  somewhat  more  abundant  in  the  form  of  small,  white,  moist 
colonies  one  to  two  millimetres  broad.  Upon  potato,  at  the  end  of  three 
days,  moist,  whitish  drops  with  ill-defined  margins. 

Pathogenesis. — Inoculations  in  the  conjunctivas  of  rabbits  produce  ecchy- 
moses  of  the  conjunctiva.  At  the  autopsy  numerous  haemorrhagic  extrava- 
sations are  found  in  all  the  organs,  especially  in  the  lungs  and  liver ;  the 
spleen  is  enlarged ;  the  bacilli  can  be  recovered  in  pure  cultures  from  the 
various  organs.  Old  cultures  proved  to  have  lost  their  virulence.  Patho- 
genic for  mice,  which  die  from  general  infection  in  the  course  of  a  few  days ; 


NOT  DESCRIBED   IN   PREVIOUS   SECTIONS. 


the  spleen  is  enlarged,  and  haemorrhages  in  the  serous  membranes  are  usually 
seen. 

BACILLUS  OF  PURPURA  H^EMORRHAGICA   OF  KOLB. 

Obtained  by  Kolb  (1891)  from  the  various  organs  of  three  individuals 
who  died  in  from  two  to  four  days  from  attacks  characterized  by  suddenly 
developed  fever,  purpura,  and  albuminous  urine. 

Morphology. — Oval  bacilli,  usually  in  pairs,  0.8  to  1.5/*long  andO.8/* 
broad,  surrounded  by  a  narrow  capsule,  which  is  only  seen  distinctly  in. 
preparations  from  the  organs. 

Stains  with  the  aniline  colors,  but  not  deeply,  and  still  more  feebly  by 
Gram's  method. 

Biological  Characters.  — An  aerobic  and  facultative  anaerobic,  non- 
liquefying  non-motile  bacillus.  Does  not  form  spores.  Grows  in  the  usual 
culture  media  at  the  room  temperature.  In  gelatin  stick  cultures,  at  the  end 
of  four  days,  a  very  small,  thin,  hyaline  growth  is  seen  about  the  point  of 
inoculation.  The  development  is  more  abundant  along  the  line  of  puncture. 
Upon  the  surface  of  agar  a  thin  layer  is  formed  with  smooth  margins. 
Upon  potato,  at  the  end  of  three  to  four  days,  a  whitish,  moist,  shining  stripe 
is  seen  along  the  impfstrich  which  is  about  three  millimetres  broad. 

Pathogenesis. — Injections  of  0.5  to  1  cubic  centimetre  of  a  bouillon 
culture  into  the  abdominal  cavity  of  rabbits  cause  symptoms  of  general  in- 


Fia.  157.  FIG.  158. 

Fi».  157.— Bacillus  gracilis  cadaveris,  from  a  gelatin  culture.  X  1,000.  From  a  photomicro- 
graph. (Steinberg.) 

FIG.  158. — Bacillus  gracilis;  colonies  in  gelatin  roll  tube,  end  of  forty -eight  hours.  X  12.  From 
a  photograph.  (Steinberg  ) 

fection  in  the  course  of  a  few  days,  and  not  infrequently  haemorrhagic  ex- 
travasations are  seen  in  the  ear  muscles.  More  than  one  cubic  centimetre 
may  cause  death  in  from  one  to  three  days.  At  the  autopsy  haemorrhagic 
extravasations  are  found  in  the  subcutaneous  tissues  and  in  the  serous  and 
mucous  membranes.  The  blood  has  little  disposition  to  coagulate;  the 
bacillus  may  be  recovered  in  pure  cultures  from  the  various  organs.  In 
guinea-pigs  local  ecchymoses  are  sometimes  produced,  otherwise  not  patho- 
genic for  this  animal.  Pathogenic  for  mice,  which  die  from  general  infec- 
tion, after  being  inoculated  with  a  small  quantity  of  a  pure  culture,  in  from 
two  to  three  days ;  spleen  enlarged;  lymphatic  glands  often  haemorrhagic. 
Not  fatal  to  dogs,  but  animals  which  were  inoculated  with  one  cubic  centi- 
metre of  a  bouillon  culture  and  subsequently  killed  proved  to  have  haemor- 
rhagic extravasations  in  the  various  organs. 


560  PATHOGENIC   AEROBIC   BACILLI 


BACILLUS   GRACILIS  CADAVERIS    (Steinberg) . 

Obtained  (1889)  from  a  fragment  of  liver,  of  man,  kept  for  forty-eight 
hours  in  an  antiseptic  wrapping. 

Morphology. — Bacilli  about  1  JLI  broad  and  2  jn  long,  associated  in  long 
chains. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile,  non-liquefying  bacillus.  Spore  formation  not  observed.  In  gelatin 
roll-tubes  the  deep  colonies  are  opaque  and  spherical ;  superficial  colonies 
circular  or  slightly  irregular  in  outline,  white  in  color,  and  opaque  or  slightly 
translucent.  In  gelatin  stab  cultures,  at  22°  C. ,  at  the  end  of  five  days  a 
rather  thick,  white  mass  at  the  point  of  puncture,  covering  one-third  of  the 
surface,  and  closely  crowded,  opaque  colonies  at  bottom  of  line  of  puncture, 
with  slender,  branching  outgrowth  above.  In  nutrient  agar,  at  the  end  of 
five  days  at  22°  C.,  a  milk-white  growth  upon  the  surface  and  opaque 
growth  to  bottom  of  line  of  puncture.  On  potato,  at  end  of  five  days  at 
22°  C.,  rather  thick,  cream-white  growth  with  irregular  margins  along  the 
impfstrich.  Cultures  in  bouillon  have  a  milky  opacity  and  a  very  disagree- 
able odor.  Grows  in  agua  coco  without  formation  of  gas. 

Pathogenic  for  rabbits  when  injected  into  the  cavity  of  the  abdomen. 

CAPSULE   BACILLUS   OF   NICOLAIER. 

Obtained  by  Nicolaier  (1894)  from  pus  contained  in  an  abscess  of  the  kid- 
ney— obtained  post-mortem. 

Morphology. — Thick  bacilli,  with  rounded  ends,  usually  four  times  as 
long  as  thick,  and  frequently  presenting  irregular  outlines  ;  often  united  in 
pairs,  and  sometimes  growing  out  into  filaments;  cocci-like  forms  also  occur. 
Often  surrounded  by  a  capsule  which  remains  unstained  in  stained  prepara- 
tions. Does  not  stain  by  Gram's  method. 

Biological  Characters. — An  aerobic,  and  facultative  anaerobic,  non- 
liquefying,  non-motile  bacillus.  Does  not  form  spores.  Grows  at  the  room 
temperature  and  more  rapidly  at  37°  C.  Upon  gelatin  plates  at  20°  C.,  at 
the  end  of  twenty-four  to  thirty-six  hours  punctiform  colonies  are  devel- 
oped, which  under  a  low  power  appear  finely  granular,  and  grayish-yellow 
spheres.  At  the  end  of  forty-eight  to  sixty  hours  the  superficial  colonies  ap- 
pear as  round  or  slightly  irregular,  grayish- white  discs,  which  project  but  lit- 
tle above  the  surface  of  the  gelatin,  and  have  thin,  transparent  margins. 
The  deep  colonies  have  a  sharply  defined  contour,  with  dark-brown  centre 
and  a  purely  granular  pale-brown  marginal  zone.  In  gelatin  stab  cultures 
a  slightly  elevated,  moist-looking,  sticky  layer  with  more  or  less  transparent 
margins  is  developed.  In  slanting  cultures  this  growth  gradually  slips  down 
to  the  lowest  part  of  the  exposed  surface,  leaving  a  thin,  gray,  transparent 
layer  over  the  gelatin  ;  along  the  line  of  puncture  a  ribbon-like,  grayish- 
white  growth  with  irregular  margins  is  developed.  In  media  containing 
glucose  some  gas  bubbles  are  developed.  The  growth  is  much  more  rapid 
in  the  incubating  oven  at  37°  C.,  and  there  is  an  abundant  development  of 
gas  in  agar  tubes.  Upon  potato  a  grayish- white,  slimy  mass  with  a  shining 
surface  is  quickly  developed.  In  bouillon,  at  the  end  of  twenty-four  hours, 
at  37°  C.,  the  medium  is  clouded  throughout,  and  a  grayish- white  deposit  ac- 
cumulates at  the  bottom  of  the  tube.  Development  occurs  also  in  acid 
media. 

Pathogenesis. — Pathogenic  for  house  mice,  white  mice,  and  for  rats — not 
for  rabbits  or  guinea-pigs — by  subcutaneous  injections.  As  Nicolaier  has 
made  a  careful  comparison  of  the  characters  of  the  various  "  capsule  bacilli" 
described,  we  quote  from  him  as  follows  : 

"Our  bacillus  in  its  morphology  and  growth  in  various  media  closely  re- 
sembles that  of  Fasching  and  of  Abel,  both  of  which  were  obtained  in  patho- 


NOT    DESCRIBED   IN   PREVIOUS   SECTIONS.  561 

logical  products  from  man.  It  is  distinguished  from  them  by  its  pathogenic 
action  upon  mice.  White  and  gray  mice  when  infected  with  our  bacillus 
die  from  septicaemia  and  show,  in  addition  to  a  serous  exudation  at  the  point 
of  inoculation,  constant  pathological  changes  in  the  kidneys,  which  may  usu- 
ally be  recognized  by  a  macroscopic  examination.  Also  by  the  spleen,  which 
is  not  always  enlarged,  and  the  liver,  which  only  in  a  few  cases  showed  any 
microscopic  changes.  In  mice  inoculated  with  the  bacillus  of  Faschmg,  or 
that  of  Abel,  which  died  of  septicaemia,  there  was  constantly  seen  an  en- 
largement of  the  spleen  (Fasching,  Abel)  and  of  the  liver  (Abel),  and  a 
cloudy  swelling  of  the  liver  and  kidneys  (Abel)  which  our  mice  failed  to 
show.  The  macroscopic  and  microscopic  changes  which  we  found  in  the 
kidneys  in  mice,  and  also  in  some  cases  in  the  liver  and  spleen,  were  not  ob- 
served by  Fasching  or  by  Abel.  Eecently  Paulseii  has  described  a  capsule  ba- 
cillus from  atrophic  rhinitis,  and  Marchand  a  capsule  bacillus — not  further 
described — which  he  obtained  in  great  numbers  from  the  exudate  in  a  case 
of  lobar  pneumonia.  Both  appear  to  be  very  similar  to  Fasching's  bacillus. 
They  are  pathogenic  for  mice,  but  do  not  cause  the  changes  in  the  kidneys 
which  we  have  described.  These  capsule  bacilli  are  therefore  not  identical 
with  ours.  Marchaiid's  bacillus  is  further  distinguished  by  the  fact  that  it  is 
pathogenic  for  guinea-pigs.  .  .  .  The  bacillus  of  Kockel  is  distinguished 
from  ours  by  the  following  characters  :  It  forms  upon  the  surface  of  gelatin, 
as  well  as  in  stick  cultures,  highly  elevated,  button  like  colonies,  while  our 
bacillus  grows  more  in  flat  and  broad  layers.  It  also  lacks  the  semi-fluid 
character  of  growth  upon  slanting  agar,  which  distinguishes  our  bacillus, 
and  as  a  result  of  which  the  growth  slips  down  to  the  lowest  point  on  the 
slanting  surface  ;  further  it  forms  upon  potato  a  yellowish  layer,  while  ours 
is  grayish- white  ;  and  it  does  not  grow  in  acid  media.  Finally,  it  is  patho- 
genic for  rabbits  by  intravenous  injection,  while  ours  is  not." 

BACILLUS   MUCOSUS   OZ^N^. 

Obtained  by  Abel  (1893)  from  cases  of  ozi^na  simplex  (rhinitis  atrophicans 
fcetida).  As  this  bacillus  appears  to  correspond  in  its  morphological  and  bio- 
logical characters  with  the  capsule  bacillus  above  described  we  shall  not 
repeat  this  description,  but  quote  from  Abel,  as  follows: 

"Thi.s  bacillus,  found  in  the  secretion  from  cases  of  ozaena,  as  the  de- 
scription we  have  given  shows,  closely  resembles  Friedlander's  pneumo- 
bacillus.  It  is  distinguished  from  it  by  certain  constant  characters.  The 
ozrena  bacillus  forms  in  cultures  a  more  fluid  mass  than  Friedlander's.  As 
a  result  of  this  it  does  not  form  the  characteristic  nail-head  culture,  but 
spreads  out  over  the  surface  of  the  gelatin.  Upon  slanting  gelatin  cultures 
the  growth  slips  down  to  the  lowest  point.  In  old  cultures  it  never  shows  a 
brown  coloring  of  the  culture  medium.  It  never  forms  gas  on  potato,  and 
in  agar  and  gelatin  cultures  but  little  gas  is  developed.  Mice  always  suc- 
cumb to  subcutaneous  inoculations,  while  Friedlander's  bacillus  does  not 
kill  mice.  Intraperitoneal  infection  of  guinea-pigs  with  the  ozseiia  bacillus 
always  causes  their  death.  Friedlander's  bacillus  only  killed  about  half  the 
guinea-pigs  inoculated  in  the  cavity  of  the  abdomen.  Finally,  Friedlander's 
bacillus  has  a  greater  tendency  to  cocci-like  forms.  The  resemblance  to 
Pfeiffers  capsule  bacillus  is  closer.  But  the  tenacious  layer  described  by 
Pfeiffer  as  found  upon  the  intestinal  coils  and  the  lungs  in  mice,  and  the 
sticky  condition  of  the  blood  and  tissue  juices  (fadenziehende)  are  want- 
ing. The  reaction  at  the  point  of  inoculation  in  mice  is  also  much  more 
pronounced  with  my  bacillus." 

It  seems  extremely  probable  that  this  bacillus,  the  Bacillus  capsulatus  mu- 
cosus  of  Fasching,  and  the  above-described  capsule  bacillus  of  Nicolaier 
are  simply  pathogenic  varieties  of  one  and  the  same  bacillus. 

36 


562  PATHOGENIC   AEROBIC   BACILLI 


CAPSULE  BACILLUS  OF  VON  DUNGERN. 

Obtained  by  von  Dungern  (1893),  post  mortem,  from  a  new-born  child 
which  died  of  hemorrhagic  septicaemia — infection  through  umbilicus. 

Morphology. — A  short,  thick  bacillus,  from  1  to  2  p.  long  and  half  as 
broad,  surrounded  by  a  capsule  which  is  slightly  stained  by  gentian  violet — 
best  seen  in  the  body  of  infected  mice  ;  sometimes  seen  in  pairs  or  in  chains 
of  four  elements  ;  also  grows  out  into  filaments,  especially  in  bouillon. 
Upon  potato  usually  only  small  spherical  elements,  resembling  micrococci, 
are  seen.  Does  not  stain  by  Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile,  non-liquefying  bacillus.  Does  not  form  spores.  Coagulates  milk, 
in  which  it  causes  an  abundant  development  of  gas  at  38°  C.  Has  feeble 
indol  reaction.  Grows  well  at  room  temperature,  more  rapidly  in  incubator. 
Upon  gelatin  plates  the  deep  colonies  at  end  of  twelve  hours  are  the  size  of  a 
pin's  head,  finely  granular,  spherical,  and  sharply  defined.  Upon  the  sur- 
face, porcelain-like,  elevated,  white  colonies  are  developed,  which  in  two  or 
three  days  attain  the  size  of  lentils.  In  gelatin  stab  cultures  development 
occurs  all  along  the  line  of  puncture,  frequently  with  formation  of  gas  bub- 
bles. Upon  agar  a  thick,  soft  layer  of  a  white  color  is  developed.  In  bouil- 
lon, at  38°  C.,  there  is  considerable  development  of  gas.  Upon  potato  the 
growth  is  very  abundant,  of  a  pale  yellowish-white  color,  thick,  soft,  some- 
what sticky,  and  filled  with  gas  bubbles.  A  great  portion  of  the  surface  is 
covered  by  this  growth  at  the  end  of  twenty -four  hours,  even  at  the  room 
temperature.  These  cultures  give  off  a  peculiar  odor,  sometimes  aromatic- 
foetid  and  sometimes  recalling  that  of  fresh  bread.  Some  of  the  cultures  on 
potato  soon  become  cream-like  in  consistence.  At  first  they  have  an  alkaline 
and  later  an  acid  reaction,  when  they  have  the  odor  of  acetic  acid. 

Pathogenesis. — Very  pathogenic  for  white  mice.  The  bacilli  are  found 
in  the  blood  and  in  all  the  organs  in  enormo'us  numbers.  At  the  point  of 
inoculation  there  is  frequently  a  hemorrhagic  oedema.  The  spleen  is  greatly 
enlarged.  Also  pathogenic  for  guinea-pigs  when  injected  into  the  cavity  of 
the  abdomen — less  pathogenic  for  rabbits. 

According  to  von  Dungern,  this  bacillus  can  not  be  distinguished  by  its 
morphological  and  biological  characters  from  Fried  lander's  bacillus,  Bacil- 
lus capsulatus  of  Pfeiffer,  or  Bacillus  canalis  capsulatus  of  Mori.  But  it  is 
distinguished  from  these  by  greater  virulence,  especially  for  rabbits,  and  by 
the  fact  that  it  frequently  gives  rise  to  hernorrliagic  extravasations  in  inocu- 
lated animals.  In  our  opinion  the  characters  given  do  not  justify  the  view 
that  this  bacillus  is  a  distinct  species  from  the  bacilli  above  mentioned. 

BACILLUS  PESTIS  (Kitasato  and  Yersin). 

Discovered  by  Kitasato  (1894)  in  the  blood  of  living  patients,  and 
in  the  buboes,  blood,  and  organs  of  those  who  had  recently  died  from 
the  infectious  malady  known  as  bubonic  plague.  Kitasato  was  sent 
to  Hong-Kong  by  the  Japanese  Government  for  the  purpose  of  inves- 
tigating this  disease.  According  to  Lowson  the  bacilli  are  found  in 
the  faeces,  in  the  contents  of  the  buboes,  and  in  the  blood. 

Morphology. — In  his  preliminary  note,  Kitasato  described  the 
plague  bacilli  as  "rods  with  rounded  ends,"  which  are  readily 
stained  by  the  ordinary  aniline  dj-es,  the  poles  being  stained  darker 
than  the  middle  part,  especially  in  blood  preparations,  and  present- 
ing a  capsule  sometimes  well  marked,  sometimes  indistinct. 


NOT  DESCRIBED   IN  PREVIOUS   SECTIONS.  563 

Yersin,  who  was  sent  by  the  French  Government  to  study  the 
bubonic  plague  at  Hong-Kong,  arrived  in  that  city  on  the  loth  of 
June,  1894.  He  describes  the  bacillus  found  in  the  contents  of  the 
buboes  as  being  short  and  thick,  with  rounded  ends,  staining  easily 
with  the  aniline  colors,  but  not  by  Gram's  method.  "  The  extremities 
stain  more  intensely  than  the  centre,  so  that  they  often  present  a 
clear  space  in  the  middle.  Sometimes  the  bacilli  appear  to  be  sur- 
rounded by  a  capsule.  ...  In  bouillon  the  bacillus  has  a  very  char- 
acteristic appearance,  resembling  the  cultures  of  the  streptococcus  of 
erysipelas — a  clear  liquid  with  grumous  deposits  on  the  walls  and  at 
the  bottom  of  the  tube.  These  cultures  examined  under  the  micro- 
scope show  veritable  chains  of  short  bacilli,  presenting  in  places  a 
considerable  spherical  enlargement." 

This  bacillus  is  sometimes  seen  to  be  motile,  and  it  has  flagella, 
which,  however,  are  difficult  to  stain  (Gordon). 

In  agar  cultures,  in  the  incubator  at  37°  C.,  involution  forms  soon 
appear.  These  may  be  spherical,  oval,  pyriform,  etc.,  and  are  often 
many  times  larger  than  the  typical  bacillus. 

Biological  Characters. — We  quote  from  Kitasato's  preliminary 
report  as  follows : 

The  bacilli  show  very  little  movement,  and  those  grown  in  the  incubator, 
in  beef -tea,  make  the  medium  somewhat  cloudy.  The  growth  of  the  bacilli  is 
strongest  011  blood  serum  at  the  normal  temperature  of  the  human  body 
(34°  C.) ;  under  these  conditions  they  develop  luxuriantly  and  form  a  col- 
ony moist  in  consistence  and  of  a  yellowish-gray  color ;  they  do  not  liquefy 
the  serum.  On  agar-agar  jelly  (the  best  is  good  glycerin  agar)  they  also 
grow  freely.  The  different  colonies  are  of  a  whitish-gray  color  and  by  re- 
flected light  have  a  bluish  appearance  ;  under  the  microscope  they  appear 
moist  aiicl  in  rounded  patches  with  uneven  edges  ;  at  first  they  appear  every- 
where as  if  piled  up  with  "glass-wool,"  later  as  if  having  dense,  large  cen- 
tres. If  a  cover-glass  preparation  is  made  from  a  cultivation  on  agar-agar, 
and,  after  having  been  stained,  is  observed  under  the  microscope,  long 
threads  of  bacilli  are  seen,  which  might,  by  careless  inspection,  be  mistaken 
for  a  coccus  chain, but  are  recognized  with  certainty  as  "  threads  of  bacilli " 
under  closer  observation.  The  growth  on  agar-gelatin  is  similar  to  that  011 
agar-agar  ;  in  a  puncture  cultivation  at  the  ordinary  temperature  after  a  few 
days  they  are  found  growing  as  a  fine  dust  in  little  points  alongside  the 
puncture,  but  with  very  little  growth  on  the  surface.  Whether  these  ba- 
cilli are  able  to  liquefy  ordinary  gelatin  or  not  I  am  at  present  unable  to  de- 
cide, as  the  temperature  of  Hong-Kong  ranges  so  high  that  the  employment 
of  simple  nutritive  gelatin  is  out  of  the  question.  I  shall  give  further  infor- 
mation on  this  question  later.  On  potatoes  at  a  temperature  of  from  28° 
to  30°  C.,  there  was  no  growth  after  ten  days'  observation,  but  at  a  tempera- 
ture of  37°  C.  the  bacilli  developed  sparingly  after  a  few  days  ;  the  growth 
was  whitish-gray  in  color  and  exsiccated.  As  mentioned  before,  the  bacilli 
grow  best  at  a  temperature  of  from  38°  to  39°  C. ;  at  how  low  a  temperature 
growth  is  possible  I  am  unable  at  present  to  state.  So  far  I  have  been  un- 
able to  observe  the  formation  of  spores. 

Experiments  on  Animals. — Mice,  rats,  guinea-pigs,  and  rabbits  are  sus- 
ceptible to  inoculation.  If  these  animals  are  inoculated  with  pure  culti- 
vations, or  with  the  blood  of  a  plague  patient  in  which  the  bacilli  have  been 


504  PATHOGENIC   AEROBIC   BACILLI 

observed,  or  with  the  contents  of  a  bubo,  or  with  pieces  of  internal  organs, 
or  even  with  the  contents  of  the  intestine,  they  begin  to  become  ill  in  from 
one  to  two  days,  according  to  the  size  of  the  animal.  Their  eyes  become  wa- 
tery, they  begin  to  show  disinclination  for  any  effort,  later  they  avoid  their 
food,  and  hide  quietly  in  a  corner  of  the  cage.  The  temperature  rises  to 
41.5°  C.,  and  with  convulsive  symptoms  they  die  in  from  two  to  five  days.  I 
must  observe  that  in  Hong-Kong  I  could  only  obtain  small  guinea-pigs 
(weight  from  one  hundred  to  one  hundred  and  fifty  grammes)  and  small 
rabbits  (from  two  hundred  to  two  hundred  and  fifty  grammes).  If  I  could 
have  experimented  upon  larger  animals  it  is  possible  that  life  would  have 
been  prolonged  somewhat  beyond  the  periods  mentioned  above.  The  parts 
around  the  point  of  inoculation  are  infiltrated  with  a  reddish  gelatinous 
exudation,  the  spleen  is  enlarged,  sometimes  there  is  a  swelling  of  the  lym- 
phatic glands,  and  in  all  the  orsrans  the  bacilli  are  found.  The  results  found 
after  death  in  animals  are  very  similar  to  those  found  in  anthrax  and  in 
oedema  malign um.  Pigeons  do  not  appear  to  be  susceptible  to  the  influence 
of  the  bacilli.  I  made  experiments  by  feeding  some  mice  and  guinea-pigs 
with  pure  cultivations  of  the  bacillus  and  with  small  pieces  of  the  internal 
organs  :  the  result  was,  such  animals  perished  in  a  few  days  under  the  same 
symptoms  as  those  which  had  been  inoculated.  In  all  the  internal  organs 
of  animals  so  destroyed  I  found  the  bacilli.  With  the  dust  of  dwelling- 
houses  from  which  the  plague-stricken  had  been  removed,  I  made  sev- 
eral experiments  upon  animals.  Some  of  the  animals  died  from  tetanus. 
In  one  case  only  a  guinea-pig  died  with  plague  symptoms,  and  in  this  ani- 
mal the  same  bacilli  were  found  in  the  internal  organs  as  in  those  of 
plague  patients  who  had  succumbed.  These  experiments  with  the  dust  from 
infected  houses  I  shall  certainly  continue.  Many  rats  and  mice  at  present 
die  spontaneously  in  Hong-Kong.  I  examined  some  of  them.  In  the  inter- 
nal organs  of  a  mouse  I  discovered  the  same  bacilli. 

Experiments  with  Desiccation, — The  contents  of  a  bubo  in  which  the 
bacilli  were  present  in  great  numbers  were  wiped  over  cover  glasses  (per- 
fectly cleansed  by  heat  and  alcohol),  'and  some  of  these  cover-glasses  were 
dried  in  the  air  of  a  room  at  a  temperature  ranging  from  28°  to  30  C.  Oth- 
ers I  exposed  directly  to  the  sun's  rays,  and  from  among  them,  after  an  expo- 
sure of  from  one,  two,  and  three  hours  up  to  six  days,  I  removed  some  parts, 
putting  such  portions  in  beef-tea  and  placing  them  in  the  incubator.  Those 
which  had  been  standing  in  the  room  from  one  to  thirty-six  hours  showed  a 
pretty  good  growth  in  the  incubator,  but  those  which  had  been  in  the  room 
for  more  than  four  days  were  unable  to  show  any  growth  even  after  one 
week's  incubation.  Those  exposed  directly  to  the  sun  were  all  destroyed  after 
from  three  to  four  hours.  Further  cultivations  011  serum  were  treated 
exactly  like  the  contents  of  the  bubo  with  very  similar  results. 

Experiment*  tn'th  Heat. — Beef -tea  cultivations  which  had  been  heated 
for  thirty  minutes  in  a  water  bath  up  to  80°  C.  were  destroyed:  at  100°  C.,  in 
the  vapor  apparatus  they  were  destroyed  in  a  few  minutes. 

Yersin  reports  that  when  fragments  of  the  spleen  or  liver  of 
animals  which  have  died  of  the  plague  are  fed  to  rats  and  mice  they 
usually  become  infected  and  die,  and  the  bacillus  is  found  in  their 
organs,  lymphatic  glands,  and  blood.  He  also  demonstrated  the  pres- 
ence of  the  bacilli  in  dead  rats  found  in  the  houses  or  streets  of 
Hong-Kong. 

Without  doubt  rats  play  an  important  part  in  the  propagation  of 
the  disease.  Monkeys  are  also  very  susceptible  to  infection,  and  it  is 
said  that  the  disease  has  been  known  to  occur  as  an  epidemic  among 
these  animals.  There  is  also  good  reason  to  believe  that  fleas  have 
some  influence  in  the  propagation  of  the  disease,  by  transferring  the 


NOT   DESCRIBED   IN   PREVIOUS   SECTIONS.  565 

bacillus  from  infected  rats  to  man,  or  from  one  individual  to  another. 
Infection  in  man  occurs  by  inoculation  through  lesions  of  the  skin 
and  also  by  the  respiratory  passages  (pulmonic  form). 

BACILLUS  PISCICIDUS  AGILIS  (Sieber) . 

Discovered  by  Sieber  (1895)  in  infected  fish,  which  died  of  an  epidemic 
disease  in  the  laboratory  of  Professor  Nencki,  at  St.  Petersburg. 

Morphology. — Short  bacilli,  often  united  in  pairs. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  motile, 
liquefying  bacillus.  In  old  cultures  in  bouillon  spores  are  developed. 
Grows  at  temperatures  of  from  12°  to  37.5°  C.  Thermal  death  point,  60  to 
65°  C.  On  gelatin  and  agar  plates  forms  granular,  grayish,  or  yellowish 
colonies,  which  appear  to  be  made  up  of  three  concentric  rings — the  outer  one 
having  a  jagged  outline.  Gas  is  developed  during  the  growth  of  the  bacillus 
—carbon  dioxide  and  methyl  merecaptan  in  small  amount.  Upon  potato  it 
forms  yellowish-brown,  pearl -like  colonies.  Causes  coagulation  of  milk. 
Ketains  its  vitality  and  virulence  for  months  in  well  or  river  water. 

Pathogenesis. — Pathogenic  for  fish,  frogs,  guinea-pigs,  rabbits,  mice, 
'and  dogs  (not  for  birds).  Old  cultures  are  more  pathogenic  than  recent 
ones,  and  gelatin  cultures  are  the  most  active.  Frogs  are  killed  in  half  an 
hour  by  0.1  cubic  centimetre  of  a  bouillon  culture  six  days  old.  Filtered 
cultures  are  as  toxic  as  those  containing  the  living  bacillus  ;  they  give  with 
iron  chloride  a  characteristic  color  reaction — an  intense  red  color.  Sieber 
has  obtained  from  his  cultures  an  extremely  toxic  alkaloid  in  the  form  of  a 
hydrochlorate.  Two  litres  of  filtered  culture  gave  0.1  gramme  of  this  salt. 
An  aqueous  solution  of  this  killed  a  frog  in.  fifteen  minutes  in  the  dose  of 
0.0035  gramme. 

BACILLUS   OF   MERES HKOWSKY. 

Obtained  by  Mereshkowsky  (1894)  from  infected  animals  (Sperniophilus 
musicus)  which  died  from  an  epidemic  malady  developed  in  his  laboratory. 

Morphology. — Closely  resembles  Loffler's  Bacillus  typhi  murium. 

Biological  Characters. — An  aerobic,  motile,  non-liquefying  bacillus. 
Spore  formation  not  observed.  Grows  in  the  usual  culture  media  at  the 
room  temperature  — best  at  37.5°  C.  In.  bouillon,  at  the  end  of  twenty -four 
hours,  the  medium  is  clouded  and  a  white  pellicle  is  seen  upon  the  surface, 
which  breaks  up  into  small  flocculi  and  falls  to  the  bottom  when  the  tube 
is  slightly  shaken.  On  gelatin  plates  minute,  slightly  granular,  pale-brown 
colonies  may  be  seen,  under  a  low  power  at  the  end  of  twenty-four  hours; 
on  the  second  day  these  are  visible  as  white  spheres,  which  under  the  micro- 
scope have  a  pale-brown  color  and  a  more  or  less  transparent,  peripheral 
zone.  In  media  containing  glucose  no  gas  is  developed.  The  growth  upon 
agar  and  potato  presents  nothing  characteristic. 

Pathogenesis. — Pathogenic  for  Zieselmausen  (Spermophilus  musicus), 
for  Spermophilus  guttatus,  for  squirrels  (Sciurus  vulgaris)  for  house  mice, 
for  field  mice  (Arvicola  arvalis).  Not  pathogenic  for  man  or  for  the  domes- 
tic animals  tested,  horse,  swine,  sheep,  fowls.  Mereshkowsky  proposes  to 
use  cultures  of  this  bacillus  for  the  extermination  of  field  mice,  which  die  in 
from  one  to  ten  days  after  being  fed  upon  biscuit  wet  with  a  bouillon  cul- 
ture. 

BACILLUS   OF   EMMERICH   AND   WEIBEL. 

Obtained  by  Emmerich  and  Weibel  (1894)  from  infected  trout  in  ponds 
belonging  to  an  establishment  for  raising  these  fish.  The  disease  appeared 
as  a  superficial  "  f  urunculosis  with  secondary  development  of  abscesses  con- 
taining bloody  pus."  Death  occurred  in  from  twelve  to  twenty  days.  The 
pustules  and  secondary  abscesses  and  blood  from  the  heart  and  various  or- 
gans contained  bacilli,  which  proved  to  be  the  cause  of  the  infectious  malady. 


5G6  PATHOGENIC    AEROBIC    BACILLI 

Morphology. — Bacilli  about  as  long1  as  the  tvplioid  bacillus,  but  not  so 
thick,  very  frequently  united  in  pairs  ;  occasionally  grows  out  into  filaments. 

Biological  Characters.— An  aerobic  and  facultative  anaerobic,  lique- 
fying, non-motile  bacillus.  Does  not  form  spores.  Thermal  death  point,  60 D 
C.  Stains  with  the  usual  aniline  colors  but  not  by  Gram's  method.  Grows 
best  at  10°  to  15°  C.  The  growth  in  gelatin  is  quite  characteristic.  At  the 
end  of  two  or  three  days,  in  gelatin  plates,  at  the  room  temperature,  small 
white  colonies  are  developed  ;  in  four  or  five  days  small  gas  bubbles  or  ex- 
cavations are  seen,  at  the  bottom  of  which  lie  the  scale-like  or  rosetta-formed 
colonies.  The  margin  of  the  colonies  is  irregular  and  later  jagged.  At 
first  the  colonies  are  grayish- white  or  yellowish,  later  brownish.  The 
superficial  colonies  have  a  peculiar  lustre.  In  gelatin  stab  cultures,  colo- 
nies develop  along  the  line  of  puncture,  which  at  first  resemble  the  growtli 
of  Streptococcus  pyogenes,  and  no  development  is  seen  on  the  surface.  At 
the  end  of  five  to  seven  days  in  place  of  the  line  of  colonies  is  seen  a  channel 
filled  with  air,  or  gas  developed  by  the  separate  colonies,  the  bubbles  from 
which  coalesce.  The  funnel  formed  in  this  way  is  somewhat  larger  above, 
and  at  the  bottom  contains  a  whitish  sediment  consisting  of  bacteria  con- 
tained in  a  few  drops  of  liquefied  gelatin.  Along  the  sides  of  the  funnel 
bubble-like  cavities  may  frequently  be  seen,  at  the  bottom  of  which  the  bac- 
teria have  accumulated.  In  bouillon  a  slight  cloudiness  is  seen  near  the 
surface,  on  the  walls  of  the  test  tube;  when  slightly  shaken  this  falls  to  the 
bottom,  leaving  the  bouillon  entirely  clear.  In  agar -agar  tubes,  a  veil- 
like  stripe  develops  along  the  line  of  puncture,  and  a  grayish-yellow,  moist 
layer,  with  irregular  outlines  upon  the  surface.  After  some  weeks  this 
acquires  a  brown  color.  No  growth  occurs  upon  potato.  No  development 
occurs  in  the  incubating  oven  at  37"  C. 

Pathogenesis. — Trout  became  infected  and  died  through  direct  infection, 
subcutaneous  or  intramuscular  inoculations,  or  through  the  addition  of  cul- 
tures to  the  water  in  which  they  were  kept,  or  by  placing  infected  fish  in  the 
same  tank  with  healthy  ones. 

BACILLUS   OF   BECK. 

Synonym. — Der  Bacillus  der  Brustseuche  beim  Kaninchen. 

Obtained  by  Beck  (1892)  from  rabbits  which  died  of  an  infectious  malady 
in  the  Institut  fur  Infectionskrankheiten,  in  Berlin, 

Morphology. — Very  small  and  slender  bacilli,  about  twice  as  long  and 
twice  as  thick  as  the  influenza  bacillus  ;  some  what  pointed  at  the  extremities ; 
show  a  tendency  to  grow  out  into  filaments. 

Biological  Characters. — An  aerobic  (strict)  non-liquefying,  non-motile 
bacillus.  Spore  formation  not  observed.  Grows  at  the  room-temperature 
and  more  vigorously  at  38J  C.  Does  not  stain  by  Gram's  method.  Thermal 
death  point,  50°  C.  (five  minutes).  Resists  desiccation,  at  the  room  tempera- 
ture, for  seventeen  days,  at  37°  C.  for  three  days. 

On  gelatin  plates,  at  the  end  of  forty-eight  hours,  small,  finely  granular, 
glass-like  colonies  are  developed  ;  older  colonies  have  a  pale-brown  appear- 
ance. In  gelatin  stab  cultures  a  granular  growth  of  a  white  color  is  seen 
along  the  line  of  puncture.  Upon  agar,  at  37°  C.,  an  abundant  development 
occurs  in  twenty -four  hours.  The  line  of  puncture  seen  from  above  is  gray- 
ish-white, by  transmitted  light  bluish  and  porcelain-like  with  a  brownish 
tint.  On  agar  plates  the  colonies  have  a  yellowish-gray  appearance ;  the 
margin  of  the  finely  granular  colonies  is  sharply  defined.  In  agar  cultures 
several  days  old  the  colonies  are  sticky  and  may  be  picked  up  as  a  compact 
mass,  or  drawn  out  into  threads.  In  bouillon,  at  37°  C.,  there  is  a  slight 
cloudiness  at  the  end  of  twenty-four  hours  ;  later  the  bouillon  is  clear  and  a 
white  sediment  is  seen  at  the  bottom  of  the  tube.  In  bouillon  cultures 
especially,  the  bacillus  grows  out  into  long  filaments. 


NOT  DESCRIBED   IN   PREVIOUS   SECTIONS.  567 

Pathogenesis. — From  0.25  to  1  cubic  centimetre  of  a  bouillon  culture 
injected  into  the  pleural  cavity  of  a  rabbit  caused  a  development  of  all  of 
the  symptoms  of  influenza  (Brustseuche) — viz.,  elevation  of  temperature  at 
the  end  of  five  or  six  hours,  cough,  nasal  discharge,  dyspnoea,  and  death— 
usually  in  from  three  to  five  days.  The  autopsy  showed  a  distinct  pleuro- 
pneumoiiia  and  a  general  blood  infection  by  the  bacillus  in  question. 
Injections  into  the  circulation  also  give  rise  to  the  symptoms  of  influenza, 
including  pneumonia,  and  to  death  at  the  end  of  from  ten  to  fourteen  days. 
Subcutaneous  injections  resulted  in  the  development  of  an  abscess  and  of  ex- 
tensive necrosis  of  the  tissues,  but  did  not  cause  a  general  blood  infection. 
Guinea-pigs  were  somewhat  less  susceptible  than  rabbits,  but  injections  into 
tlie  pleural  cavity  produced  similar  symptoms  and  death  at  a  later  date. 
White  mice  and  house  mice,  as  a  result  of  intraperitoneal  injections,  died 
within  two  or  three  days  from  general  blood  infection. 

BACILLUS  PISCICIDUS  (Fischel  and  Enoch). 

Obtained  by  Fischel  and  Enoch  (1892)  from  an  infected  carp. 

Morphology. — Bacilli  solitary  or  in  chains  of  four  to  five  elements,  1.2 
to  3  /i  long  and  0.25  ^  thick.  Stains  by  the  usual  aniline  colors  and  by 
Gram's  method. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  non- 
motile,  liquefying  bacillus.  Forms  spores.  In  gelatin  plates  forms  round 
colonies  of  a  pale  yellowish-brown  color,  having  a  slightly  toothed  border 
and  a  granular  surface.  At  the  end  of  twenty-four  hours  a  narrow  zone  of 
liquefaction  can  be  discerned  around  the  colonies,  and  at  the  end  of  about 
ten  days  the  gelatin  is  entirely  liquefied.  In  gelatin  stab  cultures  a  scanty 
growth  is  seen  along  the  line  of  inoculation  at  the  end  of  twelve  hours  ;  the 
growth  upon  the  surface  is  rapid,  and  liquefaction  commences  at  the  end  of 
twenty-four  hours.  Upon  agar,  at  37°  C.  at  the  end  of  eighteen  hours,  a  thin 
granular  layer  is  seen,  which  consists  of  small,  pale-gray  colonies.  In  agar 
stick  cultures  a  scanty  growth  occurs  along  the  line  of  puncture,  which  does 
not  increase  after  thirty-six  hours.  Upon  the  surface  the  growth  is  abundant, 
forming,  at  the  end  of  five  days  a  tolerably  thick  grayish-white  layer.  No 

frowth  occurs  upon  potato  at  the  room  temperature,  but  at  37°  C.  a  tolera- 
ly  thick,  sticky  layer  of  a  grayish- white  color  is  developed  in  three  or  four 
days.  In  bouillon,  at  37°  C.,  the  medium  is  clouded  at  the  end  of  twelve 
hours,  and  a  thin  pellicle  is  seen  upon  the  surface  at  the  end  of  thirty-six 
hours  ;  this  falls  to  the  bottom  when  the  tube  is  slightly  agitated.  At  the 
end  of  four  days  development  has  ceased,  and  the  bouillon  is  again  transpar- 
ent, while  a  flocculent  deposit  is  seen  at  the  bottom  of  the  tube.  The  bouillon 
gives  off  a  penetrating  odor,  like  that  of  burnt  milk.  The  same  odor  is  given 
off  from  cultures  in  milk,  which  is  peptonized  by  the  action  of  the  bacillus. 
At  the  end  of  twenty  days,  at  37°  C. ,  the  entire  contents  of  the  tube  have  be- 
come transparent. 

Pathogenesis. — Produces  a  fatal  infectious  disease  in  fish  ("gold  carp") 
when  inoculated  beneath  the  skin  ;  also  pathogenic  for  mice  and  for  guinea- 
pigs. 

BACILLUS   PYOGENES   FILIFORMIS   (Flexner). 

Obtained  by  Flexner  (1895)  from  the  interior  of  the  uterus  and  from  an 
exudate  in  the  pericardial  and  pleural  cavities,  of  a  rabbit  which  died  on  the 
fifth  day  after  parturition. 

Morphology. — Pleomorphous  cocci-like  forms,  short  or  long  bacilli,  and 
long  threads  are  seen  in  cover  slips  prepared  from  the  exudate.  "Very  few 
of  the  bacilli  stain  regularly  ;  for  the  most  part  brightly  stained  spots  appear 
between  stained  areas.  An  outer  membrane  always  stains,  enclosing  the 


568  PATHOGENIC   AEROBIC   BACILLI 

stained  dots  in  a  colorless  ground.     The  threads,  as  a  rule,  present  delicate, 
sinuous,  and  wavy  outlines  ;  the  short  forms  are  straight  with  rounded  ends." 

Biological  Characters. — All  attempts  to  cultivate  this  bacillus  in  the 
usual  media,  either  in  the  presence  of  oxygen  or  in  an  atmosphere  of  hydro- 
gen, proved  unsuccessful.  But  successive  cultures  were  made  by  inoculations 
in  the  pleural  cavity  of  rabbits — a  bit  of  pleural  exudate  suspended  in  bouil- 
lon was  used  for  this  purpose.  The  bacillus  was  also  propagated  upon  the 
lungs,  heart,  uterus,  and  kidney  of  healthy  rabbits.  The  organs  were  re- 
moved with  great  care  to  prevent  contamination  and  placed  in  sterilized  test 
tubes.  Transplantations  from  these  cultures  were  only  successful  for  one  or 
two  generations.  Better  results  were  obtained  ty  cultivating  the  bacillus 
upon  the  one-third  to  one-half  grown  foetuses  of  rabbits. 

Pathogenesis. — "Considerable  variations  were  observed  according  as  the 
inoculations  were  made  into  the  pleural  cavity,  the  peritoneal  cavity,  the  sub- 
cutaneous tissue,  beneath  the  dura  mater,  or  directly  into  the  circulation. 
The  inoculations  gave  positive  results  in  all  cases  except  a  few,  in  which  they 
were  made  subcutaneously.  The  death  of  the  animal  occurred  soonest  when 
inoculation  was  made  beneath  the  dura  mater.  A  small  portion  of  the  skull 
was  trephined,  care  being  taken  to  exclude  extraneous  microorganisms,  and 
a  drop  of  the  pleural  fluid  or  a  speck  of  the  fibrinous  exudate  was  introduced 
beneath  the  membranes,  care  being  taken  not  to  injure  the  brain.  These 
animals,  which  quickly  recovered  from  the  effects  of  the  operation,  died  on 
an  average  about  twelve  hours  after  the  inoculation.  .  .  . 

"  The  pleural  inoculations  were  followed  by  death,  as  before  stated,  in  ev- 
ery instance,  the  death  of  the  animal  occurring  upon  the  third  or  fourth  day. 
The  appearances  presented  at  the  autopsy  were  for  the  most  part  an  exact 
reproduction  of  those  observed  in  the  animal  which  had  succumbed  to  the 
natural  disease.  Upon  the  side  of  inoculation  a  thick,  grayish-yellow,  shaggy 
membrane  covered  the  pleural  surfaces,  being  at  times  four  or  five  millime- 
tres in  thickness.  The  pleural  cavity  contained  several  cubic  centimetres  of 
a  clear  haemoglobin-colored  fluid,  the  lung  for  the  most  part  being  com- 
pressed. At  times  smaller  or  larger  areas  of  lobular  pneumonia  would  be 
present ;  and,  as  a  rule,  the  inflammation  was  not  limited  to  the  serous  mem- 
brane of  the  side  of  inoculation,  but  extended  into  the  opposite  pleural  cavity 
and  into  the  pericardial  sac.  However,  in  these  situations  the  process  was, 
as  a  rule,  less  intense,  the  solid  exudate  being  less  considerable,  and  in  the 
case  of  the  opposite  pleural  cavity  sometimes  entirely  wanting.  The  super- 
ficial vessels,  however,  were  injected  and  the  serous  surface  of  the  affected 
membrana  covered  with  a  slimy,  clear  fluid.  In  addition  to  this  the  oppo- 
site pleural  cavity  always  contained  a  similar  pink  serum  to  that  described 
upon  the  side  of  inoculation. 

' '  The  study  of  the  exudate  upon  the  side  of  inoculation  as  well  as  the 
fluid  contained  in  the  opposite  pleural  cavity  and  in  the  pericardium  showed 
the  same  organisms  as  had  been  introduced." 


BACILLUS   DYSENTERIC. 

The  researches  of  Shiga,  of  Flexner,  and  of  the  board  of  medical 
officers  of  the  army  engaged  in  the  study  of  tropical  diseases  in  the 
Philippine  Islands  (1890)  give  support  to  the  belief  that  there  is  a 
form  of  acute  dysentery  which  is  due  to  infection  by  the  bacillus  of 
Shiga,  which  Flexner  describes  as  follows : ' 

"Bacillus  of  the  average  size  of  B.  coli  communis.  There  is  variation  in 
length :  almost  none  in  thickness.  The  individuals  are  usually  separate ; 


Johns  Hopkins  Hospital  Bulletin,  vol.  xi.,  No.  115. 


NOT   DESCRIBED    IX   PREVIOUS   SECTIONS.  569 

sometimes  they  are  united  in  pairs,  but  only  very  rarely  do  they  occur  as 
filaments.  The  ends  are  slightly  rounded.  The  bacillus  shows  moderate 
motility  :  Gram's  stain  is  negative.  . 

"  Growth  takes  place  upon  all  culture  media  at  the  room  temperature, 
but  better  in  the  thermostat.  Gelatin  is  not  liquefied.  The  colonies  resemble 
those  of  B.  typhosus,  being  more  nearly  like  them  when  first  isolated  from 
the  dejecta  than  after  a  period  of  cultivation  outside  the  body.  After  many 
months  of  such  saprophytic  growth  the  colonies  become  thicker,  exhibit  a 
moist  surface,  and  are  less  translucent.  The  strokes  upon  agar  slants  show  a 
similar  alteration.  At  first  the  growth  extends  but  little  laterally,  but  later 
on  it  becomes  two  to  three  millimetres  in  width,  and  generally  shows  distinct 
indentations  at  the  edges.  Upon  gelatin  the  colonies  are  more  delicate  ;  the 
stab  extends  along  the  line  of  puncture  only,  spreading  very  little  at  the  sur- 
face of  the  medium. 

"On  potato,  growth  takes  place  along  the  line  of  inoculation  and  spreads 
beyond.  After  some  days  it  is  a  little  elevated  and  of  a  pale-brown  tint.  On 
unfavorable  potatoes  the  growth  is  slight,  moist,  and  membranous,  resem- 
bling, except  for  the  greater  amount  of  moisture,  that  of  B.  typhosus  when 
typical. 

"  Sugars — glucose,  lactose,  and  saccharose — are  not  fermented  gaseously. 
In  glucose  media  a  moderate  acid  production  takes  place. 

"  Bouillon  is  clouded  diffusely  and  a  sediment  forms.  There  is  no  pro- 
duction of  a  pellicle. 

"  Litmus  milk  assumes,  after  twenty-four  to  seventy-two  hours,  a  faint 
lilac  tinge.  After  the  lapse  of  from  six  to  eight  days  alkali  begins  to  be  pro- 
duced, which  increases  in  amount  until  the  litmus  is  rendered  deep  blue  in 
color.  No  coagulation  of  the  milk  ensues. 

"Indol  is  not  always  formed.  Even  in  sugar-free  bouillon  it  may  fail  to 
appear,  or  it  may  be  produced  in  small  quantities  only. 

"Suitable  cultures  of  this  organism,  when  tested  for  the  agglutination  re- 
action with  the  blood  serum  of  persons  suffering  from  dysentery — the  host  of 
another  individual — give,  in  many  cases,  a  positive  result. 

"The  bacillus  is  pathogenic  for  the  ordinary  laboratory  animals.  It  is 
abundant  in  the  acute  cases  in  which  it  may  be  the  predominating  organism ; 
it  becomes  more  difficult  to  find  as  the  cases  progress  toward  recovery  or 
chronicity.  In  the  ordinary  chronic  dysentery  of  Manila,  in  which  amoebae 
are  commonly  encountered,  it  was  not  found.  It  can  be  cultivated  from  the 
dejecta  during  life,  and  the  intestinal  contents,  mucous  membrane,  and 
mesenteric  glands  in  fatal  cases. 

"Since  the  publication  of  Shiga's  studies,  Escherich  and  Celli  have  both 
attempted  to  show  that  the  organisms  obtained  from  their  respective  epidemics 
of  dysentery  are  identical  with  the  B.  dysenteriae.  In  both  cases  they  have 
proceeded  upon  the  false  assumption  that  Shiga's  microorganism  was  a 
variety  of  B.  coli  communis,  whereas,  in  point  of  fact,  it  is  much  more  nearly 
related  in  its  cultural  and  physiological  properties  to  B.  typhosus. 

"The  question  naturally  arises,  In  what  ways  does  it  differ  from  B. 
typhosus?  Comparison  of  the  Eberth-Gaffky  and  Shiga  bacilli  show  the 
criteria  of  difference  to  be  by  no  means  numerous.  The  main  features,  how- 
ever, are  as  follows  :  The  latter  shows  less  marked  motility  when  first  iso- 
lated and  a  tendency  to  lose  motility  rapidly  in  artificial  cultivations ;  it 
displays  a  more  uniform  generation  of  indol ;  after  a  brief  preliminary  acid 
production  in  milk  it  gives  rise  to  a  gradually  increasing  alkalinization  ;  it  is 
inactive  to  blood  serum  from  typhoid  cases ;  but  reacts  with  serum  from 
dysenteric  cases  to  which  B.  typhosus  does  not  respond.  .  .  . 

' '  Bearing  directly  upon  these  considerations  are  the  results  of  Lieutenant 
Strong's  studies,  continued  after  our  departure  from  Manila.  He  writes : 
'  After  you  left  we  had  a  large  number  of  acute  cases  of  dysentery.  It  seems 
certain  that  this  form,  which  we  have  begun  to  speak  of  as  acute  infectious 
dysentery,  is  independent  of  amcebae.  I  have  now  records  of  fourteen  cases 


570 

(not  all  were  fatal)  which  I  studied  bacteriologically.  From  the  stools  in  all 
of  these,  there  has  been  obtained  a  bacillus  which  agrees  with  the  organism 
obtained  by  you.  I  have  also  obtained  the  organisms  from  the  mesenteric 
glands  in  three  fatal  cases.  In  one  case  of  acute  dysentery  with  secondary 
acute  fibrinous  peritonitis  I  obtained  it  from  the  exudate.  The  agglutina- 
tion reaction  is  not  invariable.  Amoebae  were  never  demonstrable  in  any  of 
these  fourteen  cases.  On  the  other  hand,  in  every  case  with  certain  anatomi- 
cal lesions  we  always  find  the  amoebae.  In  some  cases  of  dysentery  in  which 
the  amoebae  were  absent  and  the  bacilli  present,  that  have  lasted  four  to  five 
weeks  (one  case  lasted  nearly  two  months)  and  then  resulted  fatally,  we  see 
a  continuation  of  the  same  process  that  is  observed  in  the  acute  fatal  cases. 
The  lesions  are  those  of  necroses  of  the  mucous  membrane  and  induration  of 
the  gut.' " 


XV. 

BACTERIA  OF  PLANT  DISEASES. 

I  SHALL  not  attempt  to  give  a  full  account  of  the  bacteria  which 
have  been  described  as  bearing  an  etiological  relation  to  various  in- 
fectious diseases  of  plants,  but  a  "  text- book  of  bacteriology  "  would 
be  incomplete  without  some  reference  to  the  best  known  of  these 
bacteria.  In  the  following  descriptions  of  species  I  have  preferred 
to  quote  largely  from  the  published  papers  of  Dr.  Erwin  F.  Smith, 
of  the  Department  of  Agriculture,  United  States,  a  recognized  au- 
thority in  the  investigation  of  plant  diseases,  rather  than  to  rewrite 
his  descriptions. 

BACILLUS   SOLANACEARUM    (Smith). 

Causes  a  bacterial  disease  of  the  tomato,  egg  plant,  and  Irish 
potato. 

"Morphology. — A  medium-sized  bacillus,  with  rounded  ends;  often  in 
pairs,  with  a  plain  constriction  ;  elliptical,  but  of  variable  size,  depending-  on 
age  of  culture  or  the  length  of  time  the  tissues  of  the  plant  have  been  occu- 
pied ;  usually  one  and  one-half  to  three  times  as  long  as  broad.  On  cover- 
glass  preparations  made  from  peptone  beef  bouillon  cultures  forty-eight 
hours  old  and  stained  with  a  watery  solution  of  methyl  violet,  many  are  1.5 
by  0.5  //,  but  these  measurements  must  not  be  taken  too  literally,  since  the  size 
depends  not  only  on  the  age  of  the  culture  but  also  on  the  kind  of  stain  em- 
ployed, i.e.,  on  whether  or  not  the  cell  wall  stains.  Organism  motile,  often 
only  sluggishly  so,  especially  when  taken  from  the  plant,  but  sometimes  very 
actively  motile,  especially  in  young  cultures.  Flagella  much  longer  than 
the  rod ;  several — exact  number  and  place  of  attachment  not  made  out 
clearly,  owing  to  imperfect  preparations  (Van  Ermengem's  method),  but  ap- 
parently arising  from  any  part  of  the  rod.  An  attempt  to  stain  them  by 
L6 filer's  method  was  unsuccessful.  No  spores  observed  either  in  the  plant 
or  in  culture  media,  but  the  search  has  not  been  continued  long  enough  to 
warrant  any  opinion  as  to  their  existence.  Zooglcea  are  formed  almost  from 
the  start  in  fluid  culture  media. 

"Symptoms  Produced  in  the  Plant. — The  first  indication  of  this  disease, 
or  at  least  the  first  one  to  attract  the  farmer's  attention,  is  the  sudden  wilting 
of  the  foliage.  This  may  occur  first  on  a  single  shoot,  but  finally  it  affects 
the  whole  plant.  Subsequently,  and  especially  if  the  plant  is  young  or  not 
very  woody,  the  stem  shrivels,  first  changing  to  a  yellowish-green  or  to  a 
muddy  green,  and  finally  to  brown  or  black.  The  vascular  bundles  become 
brown  long  before  the  shrivelling  takes  place,  and  in  the  potato  often  show 
through  the  outer  green  parts  of  the  stem  as  long,  dark  streaks,  or  the  bac- 
teria run  out  on  the  petioles,  after  the  manner  of  pear  blight,  forming  nar- 
row, black  lines.  The  vessels  of  such  bundles  are  filled  with  the  bacilli, 


572  BACTERIA    OF   PLANT   DISEASES. 

which  ooze  out  when  the  stein  is  cut  across.  The  foliage  may  wilt  with  or 
without  a  preliminary  yellowing1.  If  the  bacteria  are  abundant  in  the  ves- 
sels of  the  stem,  the  wilt  is  often  very  sudden  and  the  foliage  has  no  time  to 
become  yellow.  The  progress  of  the  disease  seems  to  be  more  rapid  in  young 
than  in  old  plants  and  in  hot  than  in  cold  weather. 

"In  the  case  of  the  potato  the  tubers  are  also  finally  attacked  and  de- 
stroyed, the  organism  reaching  them  by  way  of  the  vascular  bundles  of  the 
stem.  A  brown  or  black  rot  ensues,  beginning  in  the  stem  end  of  the  tuber 
in  the  vascular  ring  and  extending  in  all  directions  therefrom.  All  stages 
of  this  rot  of  the  tubers  (both  in  1895  and  in  1896)  were  obtained  repeatedly 
from  pure  cultures  of  the  bacillus  pricked  into  the  stem  several  feet  above 
ground. 

"  Bouillon  and  Peptone  Cultures. — This  organism  grows  well  at  room 
temperatures  of  20°  to  30°  C.,  in  beef  broth  peptoiiized  (Witte's  peptonum 
siccum).  It  seemed  to  make  little  difference  whether  the  bouillon  was  left 
acid  or  rendered  slightly  alkaline  with  carbonate  of  soda.  The  gathering  of 
the  zooglcea  in  the  upper  layers  of  the  fluid  is  very  distinct,  especially  if  the 
tubes  are  left  undisturbed  in  an  upright  position  for  some  days.  On  shaking 
the  turbidity  becomes  uniform.  The  organism  produces  a  copious,  dirty 
white  precipitate  (much  more  precipitate  than  B.  tracheiphilus). 

"The  inoculated  tubes  of  litmus  milk  developed  no  acid — i.e.,  showed 
no  trace  of  reddening.  After  two  or  three  days  the  litmus  became  perceptibly 
bluer  than  in  the  control  tubes,  and  this  bluing  increased  from  day  to  day, 
indicating  a  progressing  alkalinity.  This  change  took  place  at  room  tem- 
peratures of  20°  to  30  C.,  and  also  in  the  thermostat  at  37°  C.  The  casein 
was  not  precipitated. 

"  Gelatin. — In  plate  cultures  of  nutrient  gelatin  the  buried  colonies  are 
circular  in  outline  (globose),  yellowish  or  brownish,  granular  (under  Zeiss 
sixteen  millimetres  objective  and  12  compensating  ocular),  and  with  well- 
defined  margins.  No  oblong  or  spindle-shaped  colonies  could  be  found. 
The  circular  outline  and  regularity  and  distinctness  of  the  margin  of  the 
colony  were  especially  noteworthy.  Whether  these  features  will  be  found 
constant  with  all  gelatins  is  a  question  yet  to  be  determined.  Occasionally, 
after  a  few  days,  a  narrow,  clear  zone  appeared  around  the  margin  of  many  of 
these  colonies  as  if  liquefaction  had  set  in.  This,  however,  did  not  progress, 
or  increased  but  very  slowly,  and  was  clearly  visible  only  under  the  compound 
microscope.  The  buried  colonies  remained  small,  as  if  requiring  more 
oxygen  than  they  were  able  to  get.  The  surface  colonies  were  circular,  thin, 
thin -edged,  smooth,  white,  and  wet-shining.  They  did  not  spread  over  the 
plate  rapidly  or  cause  any  liquefaction  (fifteen  per  cent  gelatin,  temperature 
20°  to  27°  C.). 

"The  organism  grew  best  in  a  gelatin  of  the  following  composition: 
Lean  minced  beef,  five  hundred  cubic  centimetres ;  distilled  water,  one  thou- 
sand cubic  centimetres ;  mixed  and  set  twenty-four  hours  in  a  cool  place  ; 
filtered  and  added  ten  grammes  of  Witte's  peptonum  siccum  and  one  hundred 
and  fifty  grammes  of  L.  and  F.  gelatin.  This  gelatin  was  clarified  with  egg 
and  rendered  alkaline  with  sodium  hydrate,  titrating  with  phenolphthalein. 
The  degree  of  alkalinity  was  between  twelve  and  fourteen  of  Mr.  Fuller's 
scale. 

"Agar. — In  poured  plates  of  nutrient  agar  the  buried  colonies  differed 
considerably  from  those  in  gelatin.  Instead  of  being  circular  with  a  very 
smooth  margin,  they  were  irregularly  round  or  even  oblong,  with  a  decidedly 
irregular  granular  margin.  These  colonies  were  brown  or  yellowish-brown 
under  sixteen  millimetres  objective  and  12  ocular.  After  some  weeks  the 
whole  body  of  the  agar  became  decidedly  brown.  No  spindle-shaped  colonies 
were  to  be  seen  The  surface  colonies  grew  rather  slowly.  They  were  dirty- 
white,  smooth,  wet-shining,  and  did  not  spread  widely  over  the  agar. 

'  *  The  behavior  on  potato  is  very  characteristic.  In  twenty-four  to  forty- 
eight  hours  (temperature  27°  to  32°  C.)  the  fluid  became  turbid  and  the  pro- 


BACTERIA   OF   PLANT   DISEASES.  573 

jecting  part  of  the  cylinder  was  covered  with  a  copious,  wet-shining  growth. 
At  first  this  growth  was  white  or  dirty  white,  but  after  some  days  (three  to 
ten)  it  became  brown,  and  finally,  in  places,  nearly  or  quite  black  (smoke 
brown  is  perhaps  the  proper  term).  The  growth  on  potato  was  not  wrinkled. 
The  substratum  and  the  fluid  in  the  bottom  of  the  tube  also  became  brown. 
The  rapidity  and  the  degree  of  pigmentation  seem  to  depend  on  the  slightly 
varying  composition  of  the  potato,  apparently  on  the  amount  of  glucose 
present.  No  gas  was  formed  in  any  of  the  many  potato  cultures.  No  acid 
was  detected  in  any  stage  of  the  growth  of  the  cultures,  not  even  when  tested 
at  the  end  of  the  first  twenty-four  hours.  The  potato  cultures,  which  were 
slightly  acid  on  the  start  (normal  acidity  of  the  tuber),  soon  became  strongly 
alkaline  to  litmus  paper.  With  Nessler's  solution  the  alkaline  potato  cultures 
gave  an  immediate,* copious,  orange-yellow  reaction,  indicating  ammonia. 
These  cultures  developed  a  peculiar  odor,  often  noticed  in  rotting  potatoes, 
but  not  specially  disagreeable.  This  odor  was  likened  by  one  person  to  the 
smell  of  sour  bran.  Its  chemical  nature  has  not  been  determined.  The 
cylinders  did  not  fall  into  pieces,  but  retained  their  shape  for  several 
weeks. 

44  Gas  Production. — No  gas  appeared  in  any  of  the  many  cultures.  The 
organism  is  not  a  gas  producer. 

"Relation  to  Oxygen. — This  bacillus  appears  to  be  strictly  aerobic.  If 
ever  facultative  anaerobic,  it  is  not  so  with  any  of  the  carbohydrates  yet 
tested. 

"Acids — No  acid  reaction  could  be  detected  in  any  stage  of  any  of  the 
cultures.  Potato  cultures  only  twenty-four  hours  old  and  which  were  acid 
on  the  start  (normal  acidity  of  the  tuber)  gave  a  decided  alkaline  reaction  to 
litmus  paper.  If  any  acid  whatever  is  formed  it  is  masked  by  the  presence 
of  alkali  and  is  not  butyric  acid. 

"Alkalies. — This  organism  is  a  very  vigorous  alkali  producer.  On 
warming  the  cultures  over  a  gas  flame  or  on  placing  the  blued  strips  of 
litmus  paper  011  a  warm  glass  plate  the  alkaline  reaction  quickly  disappears. 
On  adding  a  few  drops  of  Nessler's  reagent,  as  already  stated,  a  copious 
orange-yellow  precipitate  is  at  once  developed.  This  would  indicate  that  at 
least  a  part  of  the  alkali  is  clue  to  ammonia.  Probably  amiiie  bases  are  also 
present. 

kk  The  bacillus  grows  well  in  the  thermostat  at  37°  C. — possibly  a  trifle 
better  than  outside  at  summer  temperatures  ranging  from  25°  to  32°  C. 
Under  either  condition  it  grows  rapidly.  It  still  grew  readily  from  bouillon 
cultures  after  several  weeks'  exposure  to  37 D  C.  (three  weeks'  exposure  in 
one  case,  seven  weeks'  exposure  in  another). 

"Pigments. — A  brown  pigment  is  formed  in  course  of  a  few  days  in  the 
host  plants  (potato,  tomato,  etc.),  and  in  culture  media  containing  grape, 
fruit,  or  cane  sugar  (nutrient  agar,  steamed  potato,  fermentation  tubes). 
This  pigment  is  soluble  in  water  and  glycerin.  It  is  insoluble  in  ethyl  alco- 
hol, ether,  chloroform,  xylol,  and  carbon  bisulphide." 

"BACILLUS  HYACINTHI  (Wakker). 

"  Pseudomonas  hyacinth!  (Wakker). — A  yellow,  rod-shaped  organism, 
multiplying  by  fission  ;  ends  rounded  ;  single,  in  pairs,  or  fours,  more  rarely 
in  the  form  of  chains  or  filaments  ;  motile  by  means  of  one  polar  flagellum. 
In  the  host  plant,  when  the  bundles  are  crowded  full  of  the  yellow  slime  and 
broken  down,  it  is  generally  0.8  to  1.2  by  0.4  to  0.6  /<.  In  alkaline  beef 
broth  or  on  agar  it  usually  "measures  1  to  2  by  0.4  to  0.6  /^.  In  old  cultures 
rich  in  sugar  it  often  grows  out  into  long,  slender  chains,  or  into  filaments 
(50  to  100  p  long)  in  which  there  are  no  distinct  septa.  Non-sporiferous.  Color 
distinctly  yellow,  but  somewhat  variable.  Chrome  yellow  to  pale  cadmium 
in  the  host  plant,  i.e.,  bright  yellow  (Ridgway's  nomenclature  of  colors). 
On  culture  media,  when  not  interfered  with  by  the  brown  pigment,  generally 


574  BACTERIA   OF   PLANT   DISEASES. 

gamboge,  chrome  yellow,  or  canary  yellow,  but  sometimes  paler.  Old 
cultures  on  some  media  darken  from  the  production  of  a  soluble,  pale-brown 
pigment.  This  feeble  brown  stain  is  best  developed  in  hyacinth  broth,  in 
potato  broth  with  peptone,  on  turnips,  on  radishes,  and  on  banana  rinds.  It 
was  not  observed  in  acid  or  alkaline  beef  broth,  on  cocoanut  flesh,  on  sugar 
beets,  in  nutrient  starch  jelly,  in  agar,  or  in  gelatin.,  with  or  without  sugar. 
This  organism  grows  readily  on  potato  cylinders  standing  in  distilled  water, 
but  it  never  becomes  copious  or  fills  the  water  with  a  solid  yellow  slime, 
owing  to  its  feeble  diastatic  action.  Potatoes  on  which  it  has  grown,  even  for 
several  months,  always  give  a  strong  starch  reaction  with  iodine.  It  behaves 
the  same  on  nutrient  sta.rch  jelly  free  from  assimilable  sugars.  It  liquefies 
nutrient  gelatin  and  Loffler's  blood  serum,  but  does  so  slowly,  and  will  not 
liquefy  gelatin  at  all  if  ten  per  cent  cane  sugar  is  added.  Growth  on 
nutrient  agar  or  nutrient  starch  jelly  is  inhibited  (unless  the  inoculation  be 
from  a  solid  culture  and  very  copious)  by  the  addition  of  ten  per  cent 
glycerol,  and  is  greatly  retarded  by  five-per-cent  glycerol;  even  two  and  a 
half  per  cent  of  glycerol  retarded  growth.  Growth  in  beef  broth  was  much 
retarded  by  the  addition  of  1.5-per-cent  sodium  chloride.  Organisms  ex- 
tremely sensitive  to  plant  acids,  including  those  of  the  hyacinth.  Aerobic  ; 
doubtfully,  if  ever,  facultative  anaerobic;  not  a  gas  producer.  Does  not 
redden  litmus  milk,  but  makes  it  bluer,  and  slowly  separates  the  casein  from 
the  whey  by  means  of  a  lab  ferment.  Produces  under  some  circumstances, 
and  slowly,  a  small  amount  of  non-volatile  acid  (slime  acid  ?)  with  various 
sugars  (grape,  cane,  etc.),  which  acid  is  frequently  obscured  by  the  moderate 
production  of  alkali.  In  the  presence  of  air  produces  an  organic  acid 
(probably  acetic)  from  ethyl  alcohol  dissolved  in  milk  or  bouillon.  Inverts 
cane  sugar,  but  apparently  without  the  intervention  of  any  enzyme.  Will 
not  grow  on  thirty-per-cent  grape-sugar  agar.  Resists  dry  air  very  well,  i.e., 
more  than  forty -eight  days  when  spread  on  cover  glasses  in  thin  layers. 

"  In  Dunham's  solution  with  methylene  blue  the  color  is  reduced  in  a 
few  days,  but  re-oxidizes  quickly  on  shaking  ;  final  color  (fifty- six  days)  bright 
blue.  In  Dunham's  solution  with  indigo  carmine  the  color  changes  to  a 
bright  blue,  which  persists  for  a  long  time  ;  final  color  yellowish.  In  Dun- 
ham's solution  with  rosolic  acid  and  enough  HC1  to  render  the  fluid  yellow- 
ish, Ps.  hyacinthi  did  not  redden  the  fluid,  but  made  it  colorless,  the  bac- 
terial precipitate  becoming  rosy  or  salmon-colored.  Produces  indol  slowly 
in  peptonized  beef  broth  and  in  peptonized  Uschinsky's  solution ;  does  not 
produce  nitrites  in  these  solutions.  Does  not  reduce  potassium  nitrate  to 
nitrite  in  peptonized  beef  bouillon.  Not  a  strong-smelling  germ.  Not 
readily  destroyed  by  its  own  decomposition  products  except  in  media  contain- 
ing alcohol. 

' '  Will  not  grow  in  the  thermostat  at  37°  C. ,  and  grows  very  feebly  011 
some  media  and  not  at  all  on  others  at  34°  to  35  C.  Optimum  temperature 
28°  to  30°  C.,  or  thereabouts.  Minimum  temperature  approximately  4°  C. 
Thermal  death  point  (ten  minutes'  exposure),  47.50  C.  ;  nearly  all  the  rods 
are  killed  at  47°  and  a  great  many  at  46.50°  C.  Did  not  grow  at  room  tem- 
perature after  six  days'  exposure  in  alkaline  beef  broth  in  the  thermostat  at 
35°  to  36.35°.  Does  not  grow  well  in  Uschinsky's  solution.  Grows  much 
better  in  Uschinsky's  solution  when  peptone  is  added  to  it.  Grows  well 
with  a  bright  yellow  color  on  cylinders  of  steamed  cocoanut  flesh,  standing 
with  one  end  in  distilled  water. 

"Pathogenic  to  hyacinths.  Enters  the  plant  through  wounds,  through 
the  blossoms,  etc.,  and  multiplies  in  the  vascular  system,  filling  the  vessels, 
especially  those  of  the  bulb,  with  a  bright  yellow  slime  consisting  of  bacteria. 
The  walls  of  the  vessels  are  destroyed  and  extensive  cavities  are  formed  in 
the  bundles.  The  parenchyma  around  the  bundles  is  also  involved,  but  only 
very  slowly,  the  organism  being  a  feeble  destroyer  of  cell  walls.  The  host 
plant  is  not  rapidly  destroyed,  a  year  or  more  being  necessary.  The  cells 
are  first  separated  by  solution  of  the  middle  lamella,  but  the  wall  itself  seems 


BACTERIA  OF   PLANT  DISEASES.  575 

finally  to  disappear.  The  cavities  contain  innumerable  bacteria  mingled 
with  fragments  of  the  dissolved  bundles  and  of  the  surrounding  parenchyma. 
"First  described  by  Dr.  J.  H.  Wakker  from  the  Netherlands,  where  it 
often  causes  serious  losses  in  the  hyacinth  gardens.  Not  known  to  occur  in 
any  other  part  of  the  world  "  (E.  F.  Smith). 

BACILLUS  CAMPESTRIS  (Pammel) . 

The  cause  of  brown  rot  in  Cruciferous  plants. 

"  Pseudomonas  campestris  (Pammel).  —  Yellow,  rod-shaped,  motile 
micro-organism.  Size  and  color  varying  according  to  substratum,  food 
supply,  etc.  Generally  0.7  to  3.0  by  0.4  to  0.5  ,«.  Coior  dull  wax  yellow  or 
canary  yellow.  Occasionally  as  bright  as  light  cadmium  or  as  pale  as  prim- 
rose yellow  (Ridgway's  color  scale).  One  polar  flagellum.  Non-sporiferous, 
so  far  as  known.  Pathogenic  for  various  Cruciferous  plants,  entering  and 
dwarfing  or  destroying  the  host  plant  through  the  vascular  system,  which 
becomes  decidedly  brown.  Aerobic  but,  so  far  as  known,  not  a  gas  or  acid 
producer,  i.e.,  not  facultative  anaerobic.  Forms  cavities  around  the  bundles 
but  seems  to  be  only  feebly  destructive  to  cellulose.  Produces  a  brown  pig- 
ment in  the  host  plant  and  on  steamed  Cruciferous  substrata,  especially  the 
turnip.  Grows  very  rapidly  on  steamed  potato  cylinders  at  room  tempera- 
tures, but  without  odor  or  the  formation  of  any  brown  pigment.  Liquefies 
gelatin.  Grows  feebly  at  7°  C.,  better  at  10a  C.,  but  still  feebly  ;  grows  well 
at  17°  to  19°  C. ;  grows  luxuriantly  at  21C  to  26'  C.  ;  grows  very  feebly  at  37D 
to  38°  C.  ;  will  not  grow  at  40°  C.  ;  and  is  killed  by  ten  minutes'  exposure  to 
51°  C.  Organism  closely  related  to  Wakker's  Bacterium  hyacinthi,  from 
which  it  differs,  so  far  as  I  have  been  able  to  observe,  chiefly  in  its  patho- 
genic properties,  its  duller  yellow  color  and  its  higher  thermal  death  point'* 
(E.  F.  Smith). 

PSEUDOMONAS   STEWARTI    (Smith). 

"A  medium-sized  rod  rounded  at  the  end  and  motile  by  means  of  one 
polar  flagellum,  size  0.5  to  0.9  by  1  to  2  //,  no  spores  observed  ;  found  in  enor- 
mous numbers  in  the  vascular  bundles  of  corn  (Zea  mays)  associated  with  a 
destructive  disease  of  which  it  is  probably  the  cause ;  color  in  the  host  plant 
and  in  culture  media  yellow  (buff  to  chrome  or  ochre,  occasionally  a  pale, 
dirty  yellow) ;  aerobic  and  facultative  anaerobic  ;  grows  in  all  ordinary  cul- 
ture media ;  bears  alkali  well  (soda)  and  plant  acids  extremely  well ;  grows 
luxuriantly  in  Uschinsky's  solution  ;  growth  enormously  stimulated  by  cane 
sugar,  grape  sugar,  and  galactose ;  growth  not  favored  by  five-per-cent  doses 
of  lactose,  maltose,  dextrin,  mannite,  or  glycerin  in  nutrient  starch  jelly ; 
diastatic  action  feeble,  i.e.,  able  to  obtain  food  from  starch  only  with  much 
difficulty ;  produces  alkalies  in  all  sorts  of  media  and  acids  in  the  "presence  of 
grape  and  cane  sugar;  reduces  litmus  slowly;  does  not  liquefy  gelatin 
(Stewart) ;  does  not  liquefy  Loftier1  s  blood  serum ;  grows  well  at  summer 
temperatures  of  25°  to  30  C.  ;  does  not  die  out  quickly  in  culture  media  ;  does 
not  produce  gas;  sensitive  to  light  (Stewart);  occurs  in  New  York  and 
Michigan  and  maybe  looked  for  in  all  parts  of  the  United  States"  (E.  F. 
Smith). 

BACILLUS   AMYLOVORUS   (Burrill). 

Described  by  Burrill  (1880)  as  the  cause  of  pear  blight.  Etiologi- 
cal  relation  to  this  disease  confirmed  by  Arthur  (1884  to  1887)  and 
by  Waite  (1891  to  1895). 

' '  Beginning  in  the  spring  the  germs  on  the  new  growth  of  the  season  first 
appear  on  the  negative  discs  of  the  blossoms.  The  bacilli  live  and  multiply 
in  the  nectar  and  are  able  to  enter  the  nectar  glands  without  a  puncture  or 


576  BACTERIA   OF   PLANT   DISEASES. 

injury,  and  thus  normally  get  inside  their  hosts.  The  distribution  from 
flower  to  flower  and  tree  to  tree  is  through  the  agency  of  insects,  mainly 
flower- visiting  source.  Infection  also  occurs  on  the  young  shoots  and  less 
frequently  on  the  fleshy  bark  through  injuries.  Insects  and  birds  are  agents 
of  distribution  and  inoculation  in  these  cases.  No  evidence  could  be  found 
that  the  germs  are  carried  by  the  wind.  The  blight  germs  usually  die  out  in 
the  twigs  which  are  blighted  and  dead,  but  in  certain  cases  the  germs  manage 
to  keep  alive  through  the  summer  by  making  slow  progress  in  the  fleshy 
bark.  Such  cases  may  succeed  in  living  over  winter.  Winter  weather  is 
favorable  to  the  longevity  on  account  of  the  moisture  and  low  temperature. 
The  cases  of  "hold  over"  blights  start  oft'  again  in  spring  and  exude  quanti- 
ties of  gummy  matter  full  of  the  bacilli.  This  is  visited  by  insects,  especially 
flies  and  wasps,  and  carried  on  to  the  newly  opened  flowers,  thus  completing 
the  life  cycle. 

' '  An  oval  rod-like  bacillus  0.6-0.8  n  by  1  to  6  //  long.  Constant  in  diameter 
but  varying  greatly  in  length.  Occurs  singly  or  in  young  cultures  in  pairs, 
chains,  or  masses.  Stains  readily  with  the  ordinary  aniline  dies  either  watery 
or  alcoholic  solutions.  Has  110  capsule,  but  is  supplied  with  several  flagella 
scattered  over  the  surface.  It  is  actively  motile.  Does  not  produce  spores. 
On  nutrient  beef  and  potato  broth  produces  first  a  strong  turbidity  and  a 
slight  granular  pellicle  on  the  surface,  which  breaks  up  and  settles  to  the 
bottom.  The  color  of  the  mass  is  milky  white  on  all  solid  media. 

"On  agar  plates  the  outside  colonies  at  ordinary  temperature  (18°  to  20°  C.) 
reach  a  diameter  of  about  one  millimetre  in  forty-eight  hours,  and  at  the  end 
of  a  week  become  five  to  six  millimetres  across.  A  temperature  of  36°  to 
37°  C.  starts  the  growth  more  promptly,  but  results  in  a  feebler  ultimate  de- 
velopment. 

"The  addition  of  malic  or  citric  acid  in  small  amounts  so  as  to  acidify 
the  agar,  increases  the  vigor  of  growth,  while  an  excess  of  alkali  diminishes 
it.  On  gelatin  made  from  the  commercial  brands  the  opposite  effect  is  pro- 
duced. Gelatin  should  be  neutral  to  phenolphthalein.  to  insure  vigorous 
development.  There  is  moderate  liquefaction  in  good  gelatin  culture.  A 
moderate  growth  is  made  on  sterile  potato  cylinders. 

"In  the  fermentation  tube  it  decomposes  sugar  without  the  formation  of 
gas.  It  is  most  vigorous  on  maltose,  the  cultures  becoming  strongly  acid, 
and  is  slightly  less  so  on  cane  sugar,  dextrose,  and  levulose.  It  is  aerobic  and 
facultative  anaerobic.  It  produces  no  pigment  or  coloring  matter  of  any  sort, 
and  no  odor.  It  does  not  decompose  starch.  Its  principal  food  consists  of 
nitrogenous  matter,  sugars,  and  probably,  to  some  extent,  certain  organic 
acids,  the  very  substances  which  occur  in  vigorous,  young,  growing  tissues 
of  the  host.  Certain  statements  formerly  made  are  now  known  to  be  erro- 
neous. 

"The  germ  mass  is  said  to  be  yellowish -white  on  potato.  This  could 
only  come  from  an  impure  culture,  as  the  true  pear-blight  germ  is  always 
white.  Gas,  in  some  places  COs,  is  said  to  be  formed.  This  never  oc- 
curs. Butyric  acid  is  said  to  be  one  of  the  products  of  its  decomposition. 
The  germ  produces  acid  but  never  butyric.  Starch  is  said  to  be  decomposed 
and  used  as  a  food,  but  so  far  we  have  never  been  able  to  demonstrate  this. 
The  germ  is  said  to  live  over  winter  in  the  soil.  In  our  search  we  have 
failed  to  find  it  in  such  places,  and  its  life  cycle  is  complete  without  it"  (Waite). 

BACILLUS   TRACHEIPHILUS    (Smith). 

The  cause  of  "wilt  "  in  various  species  of  Cucurbitacece — cucum- 
bers and  melons. 

"Bacilli,  often  two  or  three  times  as  long  as  broad,  of  medium  size;  soli- 
tary or  in  pairs,  occasionally  in  chains  of  four.  The  dimensions  vary  greatly 
in  the  infected  plant ;  many  rods  are  1.2  to  2.5  //  long  by  0.5  to  0.7  p  broad 


BACTERIA    OF   PLANT   DISEASES.  577 

In  cultures  the  dimensions  vary  still  more.  In  recent  cultures  the  bacilli 
exhibit  active  movements,  which  are  soon  lost.  The  bacilli  are  often  asso- 
ciated in  viscous  masses,  forming-  milk-white  drops,  which  when  touched 
with  a  platinum  needle  may  be  drawn  out  into  long  threads.  This  viscosity 
appears  to  be  due  to  a  swollen  and  partially  liquefied  capsule,  which  may  be 
demonstrated  under  the  microscope  in  stained  or  unstained  preparations. 
Does  not  form  spores.  Grows  in  bouillon,  Dunham's  solution,  etc.  Does 
not  form  a  surface  film  or  a  deposit  at  the  bottom  of  the  test  tube,  but  the 
culture  medium  is  slightly  clouded.  Grows  very  slowly  or  not  at  all  in  gela- 
tin and  does  not  liquefy.  Upon  agar-agar  it  grows  as  a  thin,  smooth,  milk- 
white,  sticky  layer,  which  extends  only  a  short  distance  from  the  point  of 
inoculation.  In  stab  cultures  it  grows  all  along  the  line  of  puncture,  form- 
ing, after  a  time,  finger-like  projections,  which  under  a  lens  are  seen  to  be 
finely  granular.  Upon  potato  it  forms  a  thin,  smooth,  white,  moist-looking 
layer,  which  only  extends  a  short  distance  from  the  line  of  inoculation. 
The  color  of  the  growth  resembles  that  of  the  potato,  and  is  much  whiter 
than  that  of  most  bacteria.  It  produces  no  pigment  and  causes  no  change 
in  the  color  of  the  potato.  In  culture  solutions  containing  dextrose,  saccha- 
rose, lactose,  or  maltose  no  gas  is  developed.  It  does  not  cause  coagulation 
of  milk.  It  grows  best  in  alkaline  media.  It  is  destroyed  by  a  temperature 
of  43°  C.  maintained  for  ten  minutes.  Cultures  in  liquid  media  or  on  potato 
usually  die  out  within  three  weeks.  It  stains  best  with  carbol-fuchsin  solu- 
tion. In  properly  stained  preparations  it  is  seen  to  have  a  capsule  and 
flagella — in  some  bacilli  one  flagellum  at  each  extremity  of  the  rod,  while  in 
others  there  are  more  "  (Smith). 

37 


XYI. 
PATHOGENIC  ANAEROBIC  BACILLI. 

STRICTLY  anaerobic  bacilli  are  not  able  to  multiply  in  the  blood 
of  living  animals  ;  but  some  of  them  may  multiply  in  the  subcuta- 
neous connective  tissue  or  in  the  muscles,  when  introduced  by  in- 
oculation, and  are  pathogenic  because  of  the  local  inflammatory  or 
necrotic  processes  to  which  they  give  rise,  or  because  they  produce 
soluble  toxic  substances  which  are  absorbed  and  cause  death  by 
their  special  action  upon  the  nervous  system  or  by  general  toxaemia. 

BACILLUS  TETANI. 

Synonyms. — The  bacillus  of  tetanus  ;  Tetanusbacillus,  Ger. 

Nicolaier  (1884)  produced  tetanus  in  mice  and  rabbits  by  intro- 
ducing garden  earth  beneath  their  skin,  and  showed  that  the  disease 
might  be  transmitted  to  other  animals  by  inoculations  with  pus  or 
cultures  in  blood  serum  containing  the  tetanus  bacillus,  which,  how- 
ever, he  did  not  succeed  in  obtaining  in  pure  cultures.  Carle  and 
Rattone  (1884)  showed  that  tetanus  is  an  infectious  disease,  which 
may  be  transmitted  by  inoculation  from  man  to  lower  animals — a 
fact  which  has  since  been  verified  by  the  experiments  of  Rosenbach 
and  others.  Obtained  in  pure  cultures  by  Kitasato  (1889). 

The  writer  produced  tetanus  in  a  rabbit  in  1880  by  injecting  be- 
neath its  skin  a  little  mud  from  the  street  gutters  in  New  Orleans. 
The  tetanus  bacillus  appears  to  be  a  widely  distributed  microorgan- 
ism in  the  superficial  layers  of  the  soil  in  temperate  and  especially  in 
tropical  regions.  In  Nicolaier's  experiments  it  was  not  found  in  soil 
from  forests  or  in  the  deeper  layers  of  garden  earth. 

Morphology. — Slender,  straight  bacilli,  with  rounded  ends, 
which  may  grow  out  into  long  filaments.  Spores  are  developed  at 
one  extremity  of  the  bacilli,  which  are  spherical  in  form  and  consid- 
erably greater  in  diameter  than  the  rods  themselves,  giving  the 
spore-bearing  bacilli  the  shape  of  a  pin. 

Stains  with  the  usual  aniline  colors  and  also  by  Gram's  method. 
The  method  of  Ziehl  may  be  employed  for  double-staining  bacilli  and 
spores. 


PATHOGENIC   ANAEROBIC   BACILLI. 


579 


Biological  Characters. — An  anaerobic,  liquefying,  motile 
bacillus.  Forms  spores.  Grows  at  the  room  temperature,  in  the 
absence  of  oxygen,  in  the  usual  culture  media.  Grows  best  at  a 
temperature  of  36°  to  38°  C.;  in  nutrient  gelatin,  at  20°  to  25°  C., 
development  is  first  seen  at  the  end  of  three  or  four  days  ;  does  not 
grow  at  a  temperature  below  14°  C.  Spores  are  formed  in  cultures 
kept  in  the  incubating  oven  at  36°  C.,  at  the  end  of  thirty  hours  ; 
in  gelatin  cultures  at  20°  to  25°  C.,  at  the  end  of  a  week  (Kitasato). 
The  bacilli  exhibit  voluntary  movements  which  are  not  very  active  ; 
those  containing  spores  are  not  motile.  It  may  be  cultivated  in  an 
atmosphere  of  hydrogen,  but  does  not  grow  in  the  presence  of  oxy- 
gen— strictly  anaerobic — or  in  an  atmosphere  of  carbon  dioxide. 
The  addition  of  one  and  one-half  to  two  per  cent  of  grape  sugar  to 
nutrient  agar  or  gelatin  causes  the  development  to  be  more  rapid 


FIG.  159. 


FIG.  160. 


FIG.  159.— Tetanus  bacillus,  from  a  gelatin  culture,  x  1,000.  From  a  photomicrograph  by 
Pfeiffer. 

FIG.  160.— Tetanusbacillus,  from  an  agar  culture  ;  spore-beating  rods.  X  1,000.  From  a  photo- 
micrograph by  Pfeiffer. 

and  abundant.     The  culture  medium  should  have  a  feebly  alkaline- 
reaction. 

Colonies  in  gelatin  plates,  in  an  atmosphere  of  hydrogen,  re- 
semble somewhat  colonies  of  Bacillus  subtilis,  the  opaque  central 
portion  being  surrounded  by  a  circle  of  diverging  rays  ;  liquefaction 
is,  however,  much  slower,  and  the  resemblance  is  lost  after  a  short 
time.  Older  colonies  resemble  the  colonies  of  certain  microscopic 
fungi,  being  made  up  of  diverging  rays.  In  long  gelatin  stab  cul- 
tures development  occurs  along  the  line  of  puncture,  at  a  consid- 
erable distance  below  the  surface,  in  the  form  of  a  radiate  out- 
growth ;  the  gelatin  is  slowly  liquefied,  and  a  small  amount  of  gas  is 
at  the  same  time  formed.  In  peptonized  bouillon  having  a  slightly 
alkaline  reaction,  under  hydrogen  gas,  the  development  is  abundant 


580 


PATHOGENIC   ANAEROBIC   BACILLI. 


and  the  cultures  give  off  a  characteristic  odor — "  brenzlichen  Ge- 
ruch  "  (Kitasato). 

According  to  Kitasato,  blood  serum  is  not  a  very  favorable  me- 
dium for  the  growth  of  the  tetanus  bacillus,  and — contrary  to  the 

statement  of  Kitt,  Tizzoni,  and  others- 
it  does  not  cause  liquefaction  of  this 
medium. 

The  spores  of  the  tetanus  bacillus  re- 
tain their  vitality  for  months  in  a  desic- 
cated condition,  and  are  not  destroyed  in 
two  and  one-half  months  when  present 
in  putrefying  material  (Turco).  They 
withstand  a  temperature  of  80°  C.  main- 
tained for  an  hour,  but  are  killed  by 
five  minutes'  exposure  to  steam  at  100°  C. 
They  are  not  destroyed  in  ten  hours  by 
a  five-per-cent  solution  of  carbolic  acid, 
but  did  not  grow  after  fifteen  hours'  ex- 
posure in  the  same  solution.  A  five- 
per-cent  solution  of  carbolic  acid,  to 
which  0.5  per  cent  of  hydrochloric  acid 
has  been  added,  destroys  them  in  two 
hours  ;  in  sublimate  solution  containing 
1  : 1,000  of  mercuric  chloride  they  are 
destroyed  at  the  end  of  three  hours,  or 
in  thirty  minutes  when  0.5  per  cent  of 
hydrochloric  acid  is  added  to  the  solu- 
tion. Kitasato  succeeded  in  obtaining 
pure  cultures  from  the  pus  formed  in 
the  vicinity  of  inoculation  wounds,  by 
destroying  the  associated  bacilli  after 
the  tetanus  bacilli  had  formed  spores. 

This  was  effected  by  heating  cultures  from  this  source  for  about  an 
hour  at  a  temperature  of  80°  C.  The  spores  of  the  tetanus  bacillus 
survived  this  exposure,  and  colonies  were  obtained  from  them  in  flat 
flasks  especially  devised  for  anaerobic  cultures  ;  from  these  colonies 
pure  cultures  in  nutrient  agar  or  gelatin — long  stick  cultures — or  in 
peptonized  bouillon  were  easily  obtained.- 

BACILLUS   CEDEMATIS   MALIGNI. 

Synonyms.—  Bacillus   of  malignant  oedema;    Yibrion  septique 
(Pasteur). 

Discovered  by  Pasteur  (1877);  carefully  studied  by  Koch  (1881). 


FIG.  161.— Culture  of  Bacillus  tetani 
in  nutrient  gelatin.    (Kitasato.) 


PATHOGENIC    ANAEROBIC   BACILLI. 


581 


This  bacillus  is  widely  distributed,  being  found  in  the  superficial 
layers  of  the  soil,  in  dust,  in  putrefying  substances,  in  the  blood  of 
animals  which  have  been  suffocated  (by  invasion  from  the  intestine), 
in  foul  water,  etc. 

It  may  usually  be  obtained  by  introducing  beneath  the  skin  of  a 
rabbit  or  a  guinea-pig  a  small  quantity  of  garden  earth.  The  animal 
dies  within  a  day  or  two,  and  this  bacillus  is  found  in  the  bloody 
serum  effused  in  the  subcutaneous  connective  tissue  for  a  consider- 
able distance  about  the  point  of  inoculation. 

Morphology. — Bacilli  from  3  to  3.5  p  long  and  1  to  1.1  /^  broad; 


FIG.   162.— Bacillus  oedematis  maligni,  from  subcutaneous  connective  tissue  of  inoculated 
guinea-pig.    X  950.    (Baumgarten.) 


frequently  united  in  pairs,  or  chains  of  three  elements  ;  may  grow 
out  into  long  filaments  15  to  40  /*  long — these  are  straight,  or  bent 
at  an  angle,  or  more  or  less  curved.  They  resemble  the  bacillus  of 

anthrax,  but  are  not  quite  as  broad,  have 
rounded  ends,,  and  in  stained  preparations 
the  long  filaments  are  not  segmented  as  is 
the  case  with  the  anthrax  bacillus.  By 
Loffler's  method  of  staining  they  are  seen  to 
have  flagella  arranged  around  the  periphery 
of  the  cells.  Large,  oval  spores  may  be  de- 
veloped in  the  bacilli  (not  in  the  long  fila- 
ments), which  are  of  greater  diameter  than 
the  rods,  and  produce  a  terminal  or  central 
swelling  of  the  same,  according  to  the  loca- 
tion of  the  spore. 

Stains  readily  by  the  aniline  colors  usu- 
ally employed,  but  is  decolorized  when  treated  by  Gram's  method. 


FIG.  163.  -Bacillus  osdema- 
tis  maligni,  from  an  agar  cul- 
ture, showing  spores.  X  1,000 
From  a  photomicrograph. 
(Frankel  and  Pfeiffer.) 


582 


PATHOGENIC   ANAEROBIC   BACILLI. 


In  stained  preparations  the  long  filaments  may  present  a  somewhat 
granular  appearance  from  unequal  action  of  the  staining  agent. 

Biological  Characters. — A  strictly  anaerobic,  liquefying,  mo- 
tile bacillus.  Forms  spores.  Grows  in  the  usual  culture  media 
when  oxygen  is  excluded — in  an  atmosphere  of  hydrogen.  Grows 
at  the  room  temperature — better  in  the  incubating  oven  at  37°  C. 
The  spores  are  formed  most  abundantly  in  cultures  kept  in  the  in- 
cubating oven,  but  may  also  be  formed  at  a  temperature  of  20°  C. 
In  the  bodies  of  animals  which  succumb  to  an  experimental  inocula- 
tion no  spores  are  found  immediately 
after  death,  but  the  bacilli  multiply  rap- 
idly in  the  cadaver,  and  form  spores 
when  the  temperature  is  favorable. 

The  malignant- oedema  bacillus  may 
be  cultivated  in  ordinary  nutrient  gela- 
tin, but  its  development  is  more  abun- 
dant when  one  to  two  per  cent  of  grape 
sugar  has  been  added  to  the  culture 
medium.  In  deep  stab  cultures  in  this 
medium  development  occurs  at  first  only 
near  the  bottom  of  the  line  of  puncture  ; 
the  gelatin  is  liquefied  and  has  a  grayish- 
white,  clouded  appearance  ;  an  abundant 
development  of  gas  occurs,  and  as  this 
accumulates  the  growth  and  liquefaction 
of  the  gelatin  extend  upward.  A  very 
characteristic  appearance  is  obtained 
when  the  bacilli  are  mixed  in  a  test 
tube  with  gelatin  which  has  been  liquefied  by  heat,  and  which  is  then 
allowed  to  solidify.  Spherical  colonies  are  developed,  in  the  course 
of  two  or  three  days,  in  the  lower  portion  of  the  gelatin  ;  these  are 
filled  with  liquefied  gelatin  of  a  grayish- white  color,  and  when  ex- 
amined with  a  low  power  are  seen  to  be  permeated  with  a  network 
of  filaments,  while  the  periphery  presents  a  radiate  appearance.  In 
nutrient  agar  growth  also  occurs  at  the  bottom  of  a  deep  punc- 
ture ;  it  has  an  irregular,  jagged  outline  and  a  granular  appearance; 
the  considerable  development  at  the  deepest  portion  and  gradual 
thinning  out  above  give  the  growth  a  club  shape  ;  in  the  incubating 
oven  there  is  an  abundant  development  of  gas,  which  often  splits  up 
the  agar  medium  and  forces  the  upper  portion  against  the  cotton 
stopper.  An  abundant  development  of  gas  also  occurs  in  cultures 
in  blood  serum,  and  the  medium  is  rapidly  liquefied  ;  at  a  tempera- 
ture of  37°  it  is  changed  in  a  few  days  to  a  yellowish  fluid,  at  the 
bottom  of  which  some  irregular,  corroded  fragments  of  the  solidified 


FIG.  164.— Bacillus  oedematis  ma- 
ligni,  cultures  in  nutrient  gelatin;  a, 
long  stab  culture :  fr,  colonies  at  bot- 
tom of  gelatin  tube.  (Flugge.) 


PATHOGENIC   ANAEROBIC    BACILLI.  583 

serum  may  be  seen.  In  agar  plates,  placed  in  a  close  receptacle 
from  which  oxygen  is  excluded,  cloudy,  dull-white  colonies  are 
formed  which  have  irregular  outlines  and  under  the  microscope 
are  seen  to  be  made  up  of  branching  and  interlaced  filaments  radi- 
ating from  the  centre.  Cultures  of  the  malignant-oedema  bacillus 
give  off  a  peculiar,  disagreeable  odor,  which  cannot,  however,  be 
designated  as  "  putrefactive." 

Pathogenesis. — Pathogenic  for  mice,  guinea-pigs,  rabbits,  and, 
according  to  Kitt,  for  horses,  dogs,  goats,  sheep,  calves,  pigs,  chick- 
ens, and  pigeons.  According  to  Arloing  and  to  Chauveau,  cattle  are 
immune.  The  disease  is  rarely  developed  except  as  a  result  of  ex- 
perimental inoculations,  but  horses  occasionally  have  malignant 
oedema  from  accidental  inoculation,  and  cases  have  been  reported 
in  man — "gangrene  gazeuse."  A  small  quantity  of  a  pure  cul- 
ture injected  beneath  the  skin  of  a  susceptible  animal  gives  rise  to 
an  extensive  inflammatory  oedema  of  the  subcutaneous  connective 
tissue  and  of  the  superficial  muscles,  which  extends  from  the  point 
of  inoculation,  especially  towards  the  more  dependent  portions  of 
the  body.  The  bloody  serum  effused  is  without  odor  and  contains 
little  if  any  gas.  But  when  malignant  oedema  results  from  the  in- 
troduction of  a  little  garden  earth  beneath  the  skin  of  a  guinea-pig  or 
other  susceptible  animal,  the  effused  serum  is  frothy  and  has  a  pu- 
trefactive odor,  no  doubt  from  the  presence  of  associated  bacteria. 
Injections  into  the  circulation  do  not  give  rise  to  malignant  oedema, 
unless  at  the  same  time  some  bacilli  are  thrown  into  the  connective 
tissue.  While  small  animals  usually  die  from  an  experimental  in- 
oculation with  a  moderately  small  quantity  of  a  pure  culture,  larger 
ones  (dogs,  sheep)  frequently  recover.  At  the  autopsy,  if  made  at 
once,  the  bacilli  are  found  in  great  numbers  in  the  effused  serum, 
but  not  in  blood  from  the  heart  or  in  preparations  made  from  the 
parenchyma  of  the  various  organs  ;  later  they  may  be  found  in  all 
parts  of  the  body  as  a  result  of  post-mortem  multiplication.  This 
applies  to  rabbits  and  to  guinea-pigs,  but  not  to  mice  ;  in  these  little 
animals  the  bacilli  may  find  their  way  into  the  blood  during  the  last 
hours  of  life,  and  their  presence  may  be  demonstrated  in  smear  prepa- 
rations of  blood  from  the  heart  or  from  the  parenchyma  of  the  spleen 
or  liver.  In  mice  the  spleen  is  considerably  enlarged,  dark  in  color, 
and  softened ;  in  rabbits  and  guinea-pigs  less  so.  With  this  excep- 
tion the  internal  organs  present  no  very  notable  pathological  changes. 

Animals  which  recover  from  malignant  cedema  are  said  to  be 
subsequently  immune  (Arloing  and  Chauveau).  Roux  and  Cham- 
berlain have  shown  that  immunity  may  be  induced  in  guinea-pigs  by 
injecting  filtered  cultures  of  the  malignant-oedema  bacillus  (about 
one  hundred  cubic  centimetres  of  a  bouillon  culture  in  three  doses) 


584 


PATHOGENIC   ANAEROBIC   BACILLI. 


into  the  abdominal  cavity ;  or,  better  still,  by  the  injection  of  fil- 
tered serum  from  animals  which  have  recently  succumbed  to  an  ex- 
perimental inoculation  (one  cubic  centimetre  repeated  daily  for  seven 
or  eight  days). 

BACILLUS  CADAVERIS. 

Obtained  by  the  writer  (1889)  from  pieces  of  liver  and  kidney,  from  yel- 
low-fever cadavers,  which  had  been  preserved  for  forty-eight  hours  in  an 
antiseptic  wrapping,  at  the  summer  temperature  of  Havana;  also  in  two 


Fio.  165.— Bacillus  cadaveris;  smear  preparation  from  liver  of  yellow-fever  cadaver,  kept 
twenty-four  hours  in  an  antiseptic  wrapping.    X  1,000.    From  a  photomicrograph.    (Sternberg.) 

cases  from  pieces  of  yellow-fever  liver  immediately  after  the  autopsy ;  also 
from  liver  preserved  in  an  antiseptic  wrapping  from  comparative  autopsies 
made  in  Baltimore. 

Morphology. — Large  bacilli  with 
square  or  slightly  rounded  corners, 
from  1.5  to  4  ^  in  length  and  about 
1.2  //  broad;  frequently  associated  in 
pairs;  may  grow  out  into  straight  or 
slightly  curved  filaments  of  from  5 
to  15  fj.  in  length. 

Biological  Characters. — An  an- 
aerobic, non-motile  bacillus ;  not 
cultivated  in  nutrient  gelatin  ;  not 
observed  to  form  spores. 

Bacillus  cadaveris  is  a  strict  anae- 
robic and  is  difficult  to  cultivate.  I 
have  succeeded  best  with  nutrient 
agar  containing  five  per  cent  of 
glycerin,  removing  the  oxygen 
thoroughly  by  passing  a  stream  of 
hydrogen  through  the  liquefied  me- 
dium. The  colonies  in  a  glycerin- 
agar  roll  tube  (containing  hydrogen 


FIG.  166.— Bacillus  cadaveris,  from  an  anae- 
robic culture  in  glycerin-agar.  X  1,000.  From 
a  photomicrograph.  (Sternberg.) 


and  hermetically  sealed)  are  opaque, 
irregular  in  outline,  granular,  and  of 


PATHOGENIC   ANAEROBIC   BACILLI. 


585 


a  white  color  by  reflected  light.  The  culture  medium  acquires  an  acid  re- 
action as  a  result  of  the  development  of  the  bacillus. 

Liver  tissue  containing  this  bacillus,  after  having  been  kept  in  an  anti- 
septic wrapping  for  forty-eight  hours,  has  a  fresh  appearance,  a  very  acid  re- 
action, and  is  without  any  putrefactive  odor. 

Pathogenesis. — Liver  tissue  containing  this  bacillus  is  rery  pathogenic 
for  guinea-pigs  when  injected  subcutaneously,  and  causes  an  extensive  in- 
flammatory cedema  extending  from  the  point  of  inoculation.  Pure  cul- 
tures of  the  bacillus  are  less  pathogenic,  and  the  few  experiments  which  I 
made  in  Havana  gave  a  somewhat  contradictory  result,  recovery  having 
occurred  in  one  guinea-pig  which  received  a  subcutaneous  injection  of  ten 
minims  of  liquid  from  an  anaerobic  culture  in  glycerin-agar,  while  another 
died  at  the  end.  of  twenty  hours  from  a  subcutaneous  injection  of  three 
minims,  with  extensive  inflammatory  cedema  in  the  vicinity  of  the  point  of 
inoculation. 


BACILLUS  OF  SYMPTOMATIC  ANTHRAX. 

Synonyms. — Rauschbrandbacillus,  Ger.  ;  Bacille  du  charbon 
symptomatique,  Fr. 

First  described  by  Bellinger  and  Feser  (1878);  carefully  studied 
and  its  principal  characters  determined  by  Arloing,  Cornevin,  and 
Thomas  (1880-83). 


FIG.  167. 


FIG.  168. 


FIG.  167.— Bacillus  of  symptomatic  anthrax,  from  an  agar  culture,  x  1,000.  From  a  photomi- 
crograph- (Frankel  and  Pfeiffer.) 

FIG.  168.— Bacillus  of  symptomatic  anthrax,  from  muscles  of  inoculated  guinea-pig.  From  a 
photomicrograph.  (Roux.) 

Found  in  the  affected  tissues  of  animals — principally  cattle — suf- 
fering from  "  black  leg,"  "  quarter  evil,"  or  symptomatic  anthrax  (Fr.  ? 
"  charbon  symptomatique";  Ger.,  "  Rauschbrand  ").  The  disease 


586 


PATHOGENIC    ANAEROBIC    BACILLI. 


prevails  during  the  summer  months  in  various  parts  of  Europe,  and 
is  characterized  by  the  appearance  of  irregular,  emphysematous 
swellings  of  the  subcutaneous  tissue  and  muscles,  especially  over  the 
quarters,  hence  the  name  "quarter  evil."  The  muscles  in  the 
affected  areas  have  a  dark  color  and  contain  a  bloody  serum  in 
which  the  bacillus  is  found. 

Morphology. — Bacilli  with  rounded  ends,  from  three  to  five  /* 
long  and  0.5  to  0.6  /*  broad  ;  sometimes  united  in  pairs,  but  do  not 
grow  out  into  filaments.  The  spores  are  oval,  somewhat  flattened  on 
one  side,  thicker  than  the  bacilli,  and  lie  near  the  middle  of  the  rods, 
but  a  little  nearer  to  one  extremity.  The  bacilli  containing  spores 
.are  somewhat  spindle-formed  (Kitasato).  "Involution  forms "  are 
quite  common  in  old  cultures  or  in  unfavorable 
media ;  in  such  cultures  variously  distorted  and 
often  greatly  enlarged  bacilli  may  be  seen,  some 
being  greatly  swollen  in  the  middle  —  spindle- 
shaped.  When  properly  stained,  by  Loffler's 
method,  a  number  of  flagella  are  seen  around  the 
periphery  of  the  cells. 

Stains  with  the  aniline  colors  usually  em- 
ployed, but  not  by  Gram's  method.  Spore-bear- 
ing bacilli  may  be  double-stained  by  first  stain- 
ing the  spores  by  ZiehFs  method,  and  then  the 
bacilli  with  a  solution  of  methylene  blue. 

Biological  Characters. — An  anaerobic,  liq- 
uefying, motile  bacillus.  Forms  spores.  Grows 
at  the  room  temperature  in  the  usual  culture  media, 
in  the  absence  of  oxygen,  in  an  atmosphere  of  hy- 
drogen, but  not  in  carbon  dioxide.  This  bacillus 
grows  more  rapidly  and  abundantly  in  nutrient 
agar  or  gelatin  to  which  1.5  to  2  per  cent  of 
grape  sugar  or  five  per  cent  of  glycerin  has  been 
added.  Colonies  in  gelatin,  in  an  atmosphere  of 
hydrogen,  are  at  first  spherical,  with  irregular  out- 
lines and  a  wart-like  surface  ;  later  the  gelatin  is 
liquefied  around  them,  and  radiating  filaments 
grow  out  into  the  gelatin,  so  that  by  transmitted 
light  they  present  the  appearance  of  an  opaque 
central  mass  with  an  irregular  surface  surrounded 
by  rays.  In  stab  cultures  in  nutrient  gelatin,  at 
20°  to  25°  C.,  at  the  end  of  two  or  three  days 
•development  occurs  at  the  bottom  of  the  line  of  puncture  to  within 
about  two  fingers'  breadth  of  the  surface  ;  the  gelatin  is  slowly 
liquefied  and  considerable  gas  is  formed.  In  old  cultures  the 


fto 


FIG.  169.— Bacillus 
of  symptomatic  an- 
thrax; long  stab  cul- 
ture in  nutrient  gela- 
tin, ten  days  at  18°- 
20°  C.  (Kitasato.) 


PATHOGENIC   ANAEROBIC   BACILLI.  587 

growth  and  liquefaction  of  the  gelatin  extend  nearly  to  the  sur- 
face. In  agar  stab  cultures,  in  the  incubating  oven,  develop- 
ment begins  within  a  day  or  two  and  extends  to  within  one 
finger's  breadth  of  the  surface ;  considerable  gas  is  evolved,  and 
the  cultures  have  a  peculiar,  acid,  penetrating  odor.  Development 
is  most  rapid  at  36°  to  38°  C.,  but  may  occur  at  a  temperature  of  16° 
to  18°  C. — not  lower  than  14°.  Spores  are  quickly  formed  in  cul- 
tures kept  in  the  incubating  oven — not  so  quickly  at  the  room  tem- 
perature. These  withstand  a  temperature  of  80°  C.  maintained  for 
an  hour,  but  are  killed  in  five  minutes  by  a  temperature  of  100°  C. 
(in  steam).  In  the  bodies  of  infected  animals  spores  are  not  formed 
until  after  the  death  of  the  animal,  at  the  end  of  twenty-four  to  forty- 
eight  hours  (Kitasato). 

The  spores  are  destroyed  by  a  five-per-cent  solution  of  carbolic 
acid  in  ten  hours,  and  the  bacilli,  in  the  absence  of  spores,  in  five 
minutes  ;  a  1  : 1,000  solution  of  mercuric  chloride  destroys  the  spores 
in  two  hours  (Kitasato).  According  to  Kitasato,  certain  shining 
bodies  of  irregular  form,  which  stain  readily  with  the  aniline  colors, 
are  to  be  seen  in  the  rods  as  they  are  found  in  the  bloody  serum  from 
an  animal  recently  dead  ;  but  these  are  not  spores,  as  some  bacterio- 
logists have  supposed. 

Pathogenesis. — Cattle,  which  are  immune  against  malignant 
oedema,  are  most  subject  to  infection  by  the  bacillus  of  symptomatic 
anthrax,  and  the  disease  produced  by  this  anaerobic  bacillus  prevails 
almost  entirely  among  them  ;  horses  are  not  attacked  spontaneously 
— i.  e. ,  by  accidental  infection — and  when  inoculated  with  a  culture  of 
this  bacillus  present  only  a  limited  local  reaction.  Swine,  dogs,  rab- 
bits, fowls,  and  pigeons  have  but  slight  susceptibility,  but  the  re- 
searches of  Aiioing,  Cornevin,  and  Thomas,  and  of  Roger  show  that 
by  the  addition  of  a  twenty-per-cent  solution  of  lactic  acid  to  a  cul- 
ture its  virulence  is  greatly  increased,  and  animals  which  have  but 
little  susceptibility,  like  the  rabbit  or  the  mouse,  succumb  to  such  in- 
jections ;  similar  results  were  obtained  by  Roger  by  the  simultaneous 
injection  of  sterilized  or  non-sterilized  cultures  of  Bacillus  prodigiosus 
or  of  Proteus  vulgaris. 

Klein  (1894)  has  obtained  from  the  spleen  of  sheep  a  bacillus 
which  corresponds  with  the  bacillus  of  malignant  oedema  in  every 
respect,  except  that  it  proved  to  be  without  pathogenic  power — "a 
non-virulent  variety  of  the  Rauschbrand  bacillus"  (Klein). 

BACILLUS   CEDEMATIS   MALIGNI  NO.    II   (Novy). 

Obtained  by  Novy  (1894)  from  the  subcutaneous  oedema  in  guinea-pigs 
which  were  inoculated  with  a  solution  of  milk-nuclein,  which  had  been  pre- 
pared from  fresh  casein. 

Morphology. — Bacilli  with  rounded  ends,  usually  solitary,  from  2.5  to 


588  PATHOGENIC   ANAEROBIC   BACILLI. 

5  //  long  and  from  0.8  to  0.9  //  broad.  Occasionally  short  and  straight  fila- 
ments, 8  to  14  ,"  long,  are  seen — very  rarely  these  reach  a  length  of  22  to  35,". 
Long  and  slender  spiral  filaments  are  found  in  pure  cultures  which  are  be- 
lieved to  be  gigantic  flagella.  These  are  seen  in  preparations  stained  with 
gentian  violet  as  unstained  spiral  filaments,  usually  from  17  to  25  j*  long  ; 
some  are  of  uniform  thickness  and  others  spindle-formed,  having  a  thickness 
of  1.7  to  2.6  p  in  the  middle,  and  tapering  to  a  scarcely  visible  line  at  the  ex- 
tremities. These  flagella  are  readily  stained  by  Loffler's  method.  They  are 
attached  to  the  periphery  of  the  rods,  as  in  the  typhoid  bacillus.  In  artifi- 
cial cultures  they  are  usually  from  40  to  50  p  long.  With  reference  to  the 
peculiar  spindle-formed  bodies  found  in  the  cultures  Novy  says  :  "  As  to  the 
character  of  these  gigantic  flagella  little  can  be  said.  Loftier,  who,  so  far  as 
I  know,  was  the  first  to  observe  these  singular  forms,  regarded  them  as  bun- 
dles or  collections  of  flagella." 

Although  at  first  inclined  to  doubt  this,  Novy  says,  in  a  postscript  to  his 
paper,  that  an  examination  of  photo-micrographs,  which  had  been  made  to 
accompany  it,  convinces  him  that  Loffler's  explanation  is  probably  correct. 

Biological  Characters. — An  anaerobic,  motile  bacillus.  The  motions  are 
not  active,  but  consist  in  a  very  moderate  to-and-fro  swinging  motion.  Does 
not  form  spores.  Does  not  grow  at  the  room  temperature.  Grows  at  tem- 
peratures of  24°  to  38°  C.  The  best  media  for  its  development  are  slightly 
alkaline  bouillon,  gelatin,  or  agar,  containing  two  per  cent  of  glucose.  May 
be  cultivated  in  a  vacuum  or  in  an  atmosphere  of  hydrogen,  carbon  dioxide, 
or  illuminating  gas.  Also  in  long  stick  cultures  in  agar.  In  glucose-agar 
plates  colonies  develop  in  fifteen  hours  at  38°  C.  These  appear  as  small, 
white  masses  the  size  of  a  pin's  head,  which,  under  the  microscope,  appear 
to  be  made  up  of  thickly  felted  threads.  The  smaller  colonies  appear  as  a 
network  of  branching  lires,  very  similar  to  the  colonies  of  the  tetanus  ba- 
cillus ;  larger  colonies  have  a  dark  centre,  with  an  irregular,  fringed  margin, 
and  are  surrounded  by  delicate  filaments.  In  glucose-agar  stab  cultures 
growth  occurs  along  the  line  of  puncture  to  within  one  cubic  centimetre  of 
the  surface,  but  is  not  as  abundant  as  the  growth  of  the  bacillus  of  malig- 
nant oedema  or  of  symptomatic  anthrax.  At  38°  C.  development  occurs  within 
twelve  to  sixteen  hours,  and  has  reached  its  maximum  at  the  end  of  twenty- 
four  hours.  An  abundant  development  of  gas  occurs,  which  splits  up  the 
agar  and  forces  the  upper  portion  towards  the  top  of  the  tube.  The  develop- 
ment of  gas  is  most  abundant  in  alkaline  media,  being  almost  absent  in  media 
having  a  neutral  or  acid  reaction.  The  most  favorable  medium  is  a  fresh  al- 
kaline bouillon  containing  two  per  cent  of  gelatin,  of  glucose,  and  of  pep- 
tone. 

Pathogenesis. — Pathogenic  for  rabbits,  guinea-pigs,  white  mice,  white  rats, 
pigeons,  and  cats.  Death  usually  results  in  from  twelve  to  thirty-six  hours 
after  the  subcutaneous  injection  of  one-tenth  to  one-fourth  cubic  centimetre 
of  a  pure  culture.  At  the  autopsy  an  extensive  subcutaneous  oedema  is  found 
extending  from  the  point  of  inoculation.  The  fluid  in  the  brawny  connective 
tissue  is  usually  colorless,  sometimes  of  a  pale-red  color.  A  small  amount  of 
gas  is  commonly  present.  The  pleural  cavities  contain  an  enormous  amount 
of  serous  exudate,  which  at  first  is  fluid,  but  when  the  autopsy  is  delayed  be- 
comes gelatinous.  In  rabbits  and  guinea-pigs  the  amount  of  this  serum  ob- 
tained from  the  pleural  cavities  may  be  from  fifty  to  sixty  cubic  centimetres. 
The  bacilli  are  usually  not  very  numerous  in  this  serum  from  the  subcuta- 
neous tissues  and  pleural  cavity. 

Kerry  (1894)  has  described  a  "  new  pathogenic  anaerobic  bacillus  "  which 
resembles  that  of  Novy  in  several  particulars.  It  does  not  grow  at  the  room 
temperature,  does  not  form  spores,  and  is  pathogenic  for  mice,  rats,  rabbits, 
and  guinea-pigs  ;  it  forms  ' '  very  long  and  thick  flagella,  which  may  be  spir- 
alig  gechlangelt."  This  bacillus  was  obtained  from  a  guinea-pig  inoculated 
with  dried  blood  (suspended  in  water  containing  lactic  acid  and  glucose) 
which  had  been  obtained  from  a  cow  that  was  supposed  to  have  died  of 
Rauschbrand. 


PATHOGENIC   ANAEROBIC   BACILLI.  589 


BACILLUS  AEROGENES   CAPSULATUS. 

Found  by  Welch  in  the  blood  vessels  of  a  patient  with  thoracic  aneurism, 
opening-  externally ;  autopsy  made  in  cool  weather  eight  hours  after  death — 
the  vessels  found  full  of  gas  bubbles. 

Morphology. — Straight  or  slightly  curved  bacilli  with  slightly  rounded 
or  sometimes  square-cut  ends ;  a  little  thicker  than  Bacillus  aiithracis,  and 
varying  in  length — average  length  3  to  6  // ;  long  threads  and  chains  are  oc- 
casionally seen.  The  bacilli,  both  from  cultures  and  in  the  animal  body,  are 
enclosed  in  a  transparent  capsule. 

Biological  Cliaracters. — An  anaerobic,  non-motile,  non-liquefying  ba- 
cillus. Does  noc  form  spores.  Grows  in  the  usual  culture  media,  in  the  ab- 
sence of  oxygen,  at  the  room  temperature,  and  produces  an  abundant  de- 
velopment of  gas  in  all.  In  nutrient  gelatin  there  is  no  marked  liquefaction, 
but  the  gelatin  is  slightly  peptonized.  In  agar,  colonies  are  developed  which 
are  usually  one  to  two  millimetres  in  diameter,  but  may  attain  a  diameter  of 
one  centimetre ;  they  are  grayish- white  in  color  and  in  the  form  of  flattened 
spheres,  ovals,  or  irregular  masses,  beset  with  little  projections  or  hair-like 
processes.  Bouillon  is  rendered  diffusely  cloudy,  with  an  abundant  white 
sediment.  Milk  is  coagulated  in  one  or  two  days.  The  cultures  in  agar  and 
bouillon  have  a  faint  odor,  comparable  to  that  of  stale  glue.  Upon  potato  a 
pale  grayish-white  layer  is  developed;  growth  occurs  at  18°  to  20°  C.,  but  is 
much  more  rapid  at  30°  to  37°  C.  Bouillon  cultures  are  sterilized  by  ex- 
posure to  a  temperature  of  58°  C.  for  ten  minutes. 

Pathogenesis. — "Quantities  up  to  2.5  cubic  centimetres  of  fresh  bouillon 
cultures  were  injected  into  the  circulation  of  rabbits  without  any  apparent 
effect,  except  in  one  instance  in  which  a  pregnant  rabbit  was  killed,  by  the 
injection  of  one  cubic  centimetre,  in  twenty-one  hours.  If  the  animal  is 
killed  shortly  after  the  injection  the  bacilli  develop  rapidly  after  death,  with 
an  abundant  formation  of  gas  in  the  blood  vessels  and  organs,  especially  the 
liver.  At  temperatures  of  18°  to  20°  C.  the  vessels,  organs,  and  serous  cavi- 
ties may  be  full  of  gas  in  eighteen  to  twenty-four  hours,  and  at  tempera- 
tures of  30°  to  32°  C.  in  four  to  six  hours,  when  one  cubic  centimetre  of  a 
bouillon  culture  has  been  injected  into  the  circulation  shortly  before  death." 

It  is  suggested  by  Welch  and  Nuttall  that  in  some  of  the  cases  in 
which  death  has  been  attributed  to  the  entrance  of  air  into  the  veins,  the  gas 
found  at  the  autopsy  may  not  have  been  atmospheric  air,  but  may  have  been 
produced  by  this  or  some  similar  microorganism  entering  the  circulation  and 
developing  after  death. 

In  a  paper  published  in  the  Bulletin  of  the  Johns  Hopkins  Hos- 
pital (September,  1900)  Professor  Welch  says:  "  Our  further  studies 
of  the  gas  bacillus  obtained  from  different  sources  have  shown  a 
moderate  range  of  variation  in  some  of  its  properties.  This  is  true 
especially  of  spore  formation,  rapidity  of  liquefaction  of  gelatin, 
presence  of  capsules,  and  virulence." 

This  bacillus  has  been  shown  by  recent  researches  to  be  widely 
distributed  in  nature,  its  natural  habitat  being  the  intestinal  canal  of 
man  and  lower  animals  and  the  soil.  It  has  considerable  importance 
in  human  pathology,  having  been  found  in  various  localized  infec- 
tious processes  in  the  subcutaneous  tissues,  the  uterus,  the  urinary 
tract,  the  liver,  the  lungs,  and  the  pleural  cavities. 


XVII. 
PATHOGENIC  SPIRILLA. 

SPIRILLUM   OBERMEIERI. 

Synonyms. — Spirochaete  Obermeieri ;  Spirillum  of  relapsing  fe- 
ver ;  Die  Recurrensspirochate. 

Discovered  by  Obermeier  (1873)  in  the  blood  of  persons  suffering 
from  relapsing  fever. 

This  spirillum  is  present,  in  very  great  numbers,  in  the  blood  of 
relapsing-fever  patients  during  the  febrile  paroxysms.  It  has  not 
been  found  under  any  other  circumstances,  and  its  etiological  rela- 
tion to  the  disease  with  which  it  is  associated  is  generally  admitted. 

Morphology. — Very  slender,  flexible,  spiral  or  wavy  filaments, 
with  pointed  ends  ;  from  sixteen  to  forty  /*  in  length  and  consider- 
ably thinner  than  the  cholera  spirillum — about  0.1  /*.  Koch  has 
demonstrated  the  presence  of  flagella  (Eisenberg). 

Stains  readily  with  the  aniline  colors,  especially  with  fuchsin, 
Bismarck  brown,  and  in  Loffler's  solution  of  methylene  blue. 

Biological  Characters. — An  aerobic,  motile  spirillum  which 
has  not  been  cultivated  in  artificial  media.  This  spirillum  appears  to 
be  a  strict  parasite,  whose  habitat  is  the  blood  of  man.  The  disap- 
pearance of  the  parasite  from  the  blood  soon  after  the  termination 
of  a  febrile  paroxysm,  and  its  reappearance  during  subsequent  par- 
oxysms, have  led  to  the  inference  that  it  must  form  spores,  but  this 
has  not  been  demonstrated.  In  fresh  preparations  from  the  blood 
the  spirillum  exhibits  active  progressive  movements,  accompanied 
by  very  rapid  rotation  in  the  long  axis  of  the  spiral  filaments,  or  by 
undulatory  movements.  The  movements  are  so  vigorous  that  the 
comparatively  large  red  blood  corpuscles  are  seen,  under  the  micro- 
scope, to  be  thrown  about  by  the  slender  spiral  filaments,  which  it  is 
difficult  to  see  in  unstained  preparations.  When  preserved  in  a  one- 
half -per-cent  salt  solution  they  continue  to  exhibit  active  movements 
for  a  considerable  time.  Efforts  to  cultivate  this  spirillum  in  artificial 
media  have  thus  far  been  unsuccessful,  although  Koch  has  observed 
an  increase  in  the  length  of  the  spirilla  and  the  formation  of  a 
tangled  mass  of  filaments. 


ERNBERO/S  BACTERIOLOGY 


Plate-    VII 


o 


Spirillurti   Obernieieri  ia  blood  of  two   monkeys, 
inoculated  after  removal  of  spleen.. 
( Soudakewil clij. 


PATHOGENIC    SPIRILLA. 


591 


In  experiments  made  by  Heydenreich  the  spirillum  was  found  to 
preserve  its  vitality  (motility)  for  fourteen  days  at  a  temperature  of 


Fio.   170.— Spirillum    Obermeieri  in  blood   of   man.     x   1,009.     From   a   photomicrograph.. 
(Frankel  and  Pfeiffer. ) 

16°  to  22°  C.,  for  twenty  hours  at  37°,  and  at  42.5°  for  two  or  three 
hours  only. 

Pathogenesis. — Causes  in  man  the  disease  known  as  relapsing 
fever.     Munch  and  Moczutkowsky  have  produced  typical  relapsing. 


FIG.  in.—Spirillum  Obermeieri  in  blood  of  an  inoculated  ape.    x  700.    (Koch. 

fever  in  healthy  persons  by  inoculating  them  with  blood  containing 
the  spirillum  of  Obermeier.  The  spirilla  are  found  in  the  blood  dur- 
ing the  febrile  paroxysm,  and  for  a  day  or  two,  at  the  outside,  after 


592  PATHOGENIC    SPIRILLA. 

its  termination  ;  sometimes  they  are  present  in  great  numbers,  and 
at  others  can  only  be  found  by  searching  several  microscopic  fields; 
they  are  not  present  in  the  various  secretions — urine,  sweat,  saliva, 
etc.  In  fatal  cases  the  principal  pathological  changes  are  found  in 
the  spleen,  which  is  greatly  enlarged,  and  in  the  liver  and  marrow 
of  the  bones,  which  contain  inflammatory  and  necrotic  foci.  Koch 
and  Carter  have  succeeded  in  transmitting  the  disease  to  monkeys 
by  subcutaneous  inoculations  with  small  amounts  of  defibrinated 
blood  containing  the  spirillum.  After  an  incubation  period  of  seve- 
ral days  typical  febrile  paroxysms  were  developed,  during  which 
the  actively  motile  spirilla  were  found  in  the  blood  in  large  numbers. 
Blood  from  one  animal,  taken  during  the  attack,  induced  a  similar 
febrile  paroxysm  when  inoculated  into  another  of  the  same  species- 
relapses,  such  as  characterize  the  disease  in  man,  were  not  observed. 
One  attack  did  not  preserve  the  animals  experimented  upon  from  a 
similar  attack  when  they  were  again  inoculated  after  an  interval  of 
a  few  days.  Soudakewitch  (1891)  has  made  successful  inoculation 
experiments  in  monkeys,  and  has  shown  that  in  monkeys  from  which 
the  spleen  has  previously  been  removed  the  spirilla  continue  to 
multiply  very  abundantly  in  the  blood  and  the  disease  has  a  fatal 
termination,  whereas  in  monkeys  from  which  the  spleen  has  not  been 
removed  the  spirilla  disappear  from  the  blood  within  a  few  days 
after  the  access  of  the  febrile  paroxysm  and  the  animal  recovers. 

SPIRILLUM   ANSERUM. 

Synonym. — Spirochseta  anserina  (Sakharoff). 

Obtained  by  Sakharoff  (1890)  from  the  blood  of  geese  affected  by  a  fatal 
form  of  septicaemia  due  to  this  spirillum.  This  disease  prevails  among  geese 
in  Caucasia,  especially  in  swampy  regions,  appearing  annually  and  destroy- 
ing a  large  number  of  the  domestic  geese. 

Morphology. — Resembles  the  spirillum  of  relapsing  fever.  The  long  and 
flexible  spiral  filaments,  when  the  disease  is  at  its  height,  are  often  seen  in 
interlaced  masses,  around  the  margins  of  which  radiate  single  filaments 
which  by  their  movements  cause  the  whole  mass  to  change  its  place,  as  if  it 
were  a  single  organism.  These  masses  are  sometimes  so  large  that  a  single 
one  occupies  the  entire  field  of  the  microscope. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters. — An  aerobic,  motile  spirillum.  Not  cultivated 
in  artificial  media.  The  movements  are  very  active,  resembling  those  of 
Spirillum  Obermeieri,  but  cease  in  an  hour  or  two  in  preparations  made  from 
the  blood  of  geese  containing  it. 

Pathogenesis.  — A  small  quantity  of  blood  from  an  infected  goose  inocu- 
lated into  a  healthy  animal  of  the  same  species  induces  the  disease  after  a 
period  of  incubation  of  four  to  five  days.  The  infected  goose  ceases  to  eat, 
becomes  apathetic,  remaining  in  one  place,  and  usually  dies  at  the  end  of  a 
week  ;  the  temperature  is  increased,  and  in  some  cases  there  is  diarrhoea. 
The  spirilla  are  found  in  the  blood  at  the  outset  of  the  malady,  but  after 
death  they  are  not  seen  either  in  the  blood  or  in  the  various  organs.  The 
heart  and  the  liver  are  found  to  have  undergone  a  fatty  degeneration,  and 
yellowish,  cheesy  granules  the  size  of  a  millet  seed  are  seen  upon  the  surface 
of  these  organs.  The  spleen  is  soft  and  easily  broken  up  by  the  fingers. 


PATHOGENIC    SPIRILLA.  593 

Inoculations  into  chickens  and  pigeons  were  without  result  ;  in  one 
chicken  the  spirilla  were  found  in  the  blood  011  the  fourth  day  after  inocula- 
tion, but  the  fowl  recovered. 

SPIRILLUM    CHOLER^E   ASIATICS. 

Synonyms. — Spirillum  ("bacillus")  of  cholera;  Comma  bacillus 
of  Koch ;  Kommabacillus  der  Cholera  Asiatica ;  Bacille-virgule 
cholerigene. 

Discovered  by  Koch  (1884)  in  the  excreta  of  cholera  patients  and 
in  the  contents  of  the  intestine  of  recent  cadavers. 

The  researches  of  Koch,  made  in  Egypt  and  in  India  (1884),  and 
subsequent  researches  by  bacteriologists  in  various  parts  of  the 
world,  show  that  this  spirillum — so-called  "  comma  bacillus  "•  —is  con- 
stantly present  in  the  contents  of  the  intestine  of  cholera  patients 
during  the  height  of  the  disease,  and  that  it  is  not  found  in  the  con- 
tents of  the  intestine  of  healthy  persons  or  of  those  suffering  from 


FIG.  172.  FIG.  173. 

FIG.  172.— Spirillum  cholerae  Asiaticae.    x  1,000.    From  a  photomicrograph.    (Koch.) 
FIG.  173.— Spirillum  cholerae  Asiaticae,  involution  forms.     X  700.    (Van  Ermengem.) 

other  diseases  than  cholera.  The  etiological  relation  of  this  spiril- 
lum to  Asiatic  cholera  is  now  generally  admitted  by  bacteriologists. 
Morphology. — Slightly  curved  rods  with  rounded  ends,  from  0.8 
to  2  n  in  length  and  about  0.3  to  0.4  /*iii  breadth.  The  rods  are 
usually  but  slightly  curved,  like  a  comma,  but  are  occasionally  in 
the  form  of  a  half-circle,  or  two  united  rods  curved  in  opposite 
directions  may  form  an  S-shaped  figure.  Under  certain  circum- 
stances the  curved  rods  grow  out  into  long,  spiral  filaments,  which 
may  consist  of  numerous  spiral  turns,  and  in  hanging-drop  cultures 
the  S-shaped  figures  may  also  be  seen  to  form  the  commencement 
of  a  spiral ;  in  stained  preparations  the  spiral  character  of  the  long 
filaments  is  often  obliterated,  or  nearly  so.  When  development  is 
very  rapid  the  short,  curved  rods  or  S-shaped  spirals  only  are  seen ; 
but  in  hanging-drop  cultures,  or  in  media  in  which  the  develop, 
38 


594 


PATHOGENIC   SPIRILLA. 


ment  is  retarded  by  an  unfavorable  temperature,  the  presence  of  a 
little  alcohol,  etc. ,  the  long,  spiral  filaments  are  quite  numerous,  and 
bacteriologists  generally  agree  that  the  so-called  "  comma  bacillus  " 
is  really  only  a  fragment  of  a  true  spirillum.  By  LofHer's  method 
of  staining  the  rods  may  be  seen  to  have  a  single  terminal  flagel- 
lum.  In  old  cultures  the  bacilli  frequently  lose  their  characteristic 
form  and  become  variously  swollen  and  distorted — involution  forms. 
Hueppe  has  described  the  appearance  of  spherical  bodies  in  the 
course  of  the  spiral  filaments,  which  he  believes  to  be  reproductive 
elements — so-called  arthrospores. 

Stains  with  the  aniline  colors  usually  employed,  but  not  as  quick- 
ly as  many  other  bacteria ;   an  aqueous  solution  of  fuchsin  is  the 


FIG.  174.  FIG.  IT."). 

Fio.  174  —Spirillum  choleree  Asiaticae;  colonies  upon  gelatin  plate,  end  of  thirty  hours.  X  100. 
Photograph  by  Frankel  and  Pfeiffer. 

Fio.  175.— Spirillum  cholerae  Asiaticse,  from  a  gelatin  culture,  x  1,000.  From  a  photomicro- 
graph. (Frankel  and  Pfeiffer.) 

most  reliable  staining  agent;  is  decolorized  by  iodine  solution- 
Grants  method.  Sections  may  be  stained  with  Loffler's  solution. 

Biological  Characters. — An  aerobic  (facultative  anaerobic), 
liquefying,  motile  spirillum.  Grows  in  the  usual  culture  media  at 
the  room  temperature — more  rapidly  in  the  incubating  oven.  Does 
not  grow  at  a  temperature  above  42°  or  below  14°  C.  Does  not  form 
endogenous  spores  (forms  arthrospores,  according  to  Hueppe  ?). 

In  gelatin  plate  cultures,  at  22°  C.,  at  the  end  of  twenty-four 
hours  small,  white  colonies  may  be  perceived  in  the  depths  of  the 
gelatin ;  these  grow  towards  the  surface  and  cause  liquefaction  of 
the  gelatin  in  the  form  of  a  funnel  which  gradually  increases  in 


PATHOGENIC   SPIRILLA. 


595 


depth,  and  at  the  bottom  of  which  is  seen  the  colony  in  the  form  of 
a  small,  white  mass  ;  as  a  result  of  this  the  plates  on  the  second  or 
third  day  appear  to  be  perforated  with  numerous  small  holes  ;  later 

the  gelatin  is  entirely  liquefied.  Under 
a  low  power  the  young  colonies,  before 
liquefaction  has  commenced,  present  a 
rather  characteristic  appearance ;  they 
are  of  a  white  or  pale-yellow  color,  and 
have  a  more  or  less  irregular  outline, 
the  margins  being  rough  and  uneven; 
the  texture  is  coarsely  granular,  and  the 
surface  looks  as  if  it  were  covered  with 
little  fragments  of  broken  glass,  while 

the  colony  has  a  shining  appearance  ;  when  liquefaction  commences  an 
ill-defined  halo  is  first  seen  to  surround  the  granular  colony,  which 
by  transmitted  light  has  a  peculiar  roseate  hue.  In  stab  cultures  in 
nutrient  gelatin  development  occurs  all  along  the  line  of  inoculation, 


FIG.  176.— Colonies  of  the  cholera 
spirillum;  a,  end  of  twenty  hours ;  6, 
end  of  thirty  hours;  c,  end  of  forty- 
eight  hours;  d,  after  liquefaction  of 
the  gelatin.  (Flugge.) 


FIG.  177.— Spirillum  choleras  Asiaticse;  a,  one  day  old;  b.  three  days  old;  c,  four  days  old;  d,  five 
days  old;  e,  seven  days  old;  /,  10  days  old.    From  photographs  by  Koch. 

but  liquefaction  of  the  gelatin  first  occurs  only  near  the  surface  ;  on 
the  second  day,  at  22°  C.,  a  short  funnel  is  formed  which  has  a 
comparatively  narrow  mouth,  and  the  upper  portion  of  which  con- 
tains air,  while  just  below  this  is  a' whitish,  viscid  mass  ;  later  the 
funnel  increases  in  depth  and  diameter,  and  at  the  end  of  from  four 
to  six  days  may  reach  the  edge  of  the  test  tube ;  in  from  eight  to 
fourteen  days  the  upper  two-thirds  of  the  gelatin  is  completely  lique- 
fied. Owing  to  the  slight  liquefaction  which  occurs  along  the  line  of 
growth  during  the  first  three  or  four  days,  the  central  mass  which 


596 


PATHOGENIC   SPIRILLA. 


had  formed  along  the  line  of  inoculation  settles  down  as  a  curled 
or  irregularly  bent,  yellowish-white  thread  in  the  lower  part  of  a 
slender  tube  filled  with  liquefied  gelatin,  the  upper  part  of  which 
widens  out  and  is  continuous  with  the  funnel  above.  Upon  the  sur- 
face of  nutrient  agar  a  moist,  shining,  white  layer  is  formed  along 
the  line  of  inoculation — impfstrich.  Blood  serum  is  slowly  liquefied 
by  this  spirillum.  Upon  the  surface  of  cooked  potato,  in  the  incu- 
bating oven,  a  rather  thin  and  semi-transparent  brown  or  grayish- 
brown  layer  is  developed.  In  bouillon  the  development  is  rapid  and 
abundant,  especially  in  the  incubating  oven  ;  the  fluid  is  only  slightly 


FIQ.  178.— Cultures  in  nutrient  gelatin,  at  the  room  temperature  (16°  to  18°  C.),  at  the  com- 
mencement of  the  fourth  day;  a,  Spirillum  cholerae  Asiaticse;  &,  Spirillum  tyrogenum ;  c,  Spirillum 
of  Finkler  and  Prior.  (Baurngarten.) 

clouded,  but  the  spirilla  accumulate  at  the  surface,  forming  a  wrin- 
kled membranous  layer.  Sterilized  milk  is  also  a  favorable  culture 
medium.  In  general  this  spirillum  grows  in  any  liquid  containing  a 
small  quantity  of  organic  pabulum  and  having  a  slightly  alkaline 
reaction.  An  acid  reaction  of  the  culture  medium  prevents  its  de- 
velopment, as  a  rule,  but  it  has  the  power  of  gradually  accommo- 
dating itself  to  the  presence  of  vegetable  acids,  and  grows  upon 
potatoes — in  the  incubator  only — which  have  a  slightly  acid  reaction. 
Abundant  development  occurs  in  bouillon  which  has  been  diluted 
with  eight  or  ten  parts  of  water,  and  the  experiments  of  Wolffhugel 


PATHOGENIC   SPIRILLA.  597 

and  Riedel  show  that  it  also  multiplies  to  some  extent  in  sterilized 
river  or  well  water,  and  that  it  preserves  its  vitality  in  such  water 
for  several  months.  But  in  milk  or  water  which  contains  other  bac- 
teria it  dies  out  in  a  few  days.  Gruber  and  Schottelius  have  shown, 
however,  that  in  bouillon  which  is  greatly  diluted  the  cholera  spiril- 
lum may  take  the  precedence  of  the  common  saprophytic  bacteria, 
and  that  they  form  upon  the  surface  of  such  a  medium  the  charac- 
teristic wrinkled  film.  Koch  found  in  his  early  investigations  that 
rapid  multiplication  may  occur  upon  the  surface  of  moist  linen,  and 
also  demonstrated  the  presence  of  this  spirillum  in  the  foul  water  of 
a  "  tank "  in  India  which  was  used  by  the  natives  for  drinking 
purposes.  In  the  experiments  of  Bolton  (1886)  the  cholera  spirillum 
was  found  to  multiply  abundantly  in  distilled  water  to  which 
bouillon  was  added  in  the  proportion  of  fifteen  to  twenty-five  parts 
in  one  thousand. 

The  thermal  death-point  of  the  cholera  spirillum  in  recent  cul- 
tures in  flesh-peptone-gelatin,  as  determined  by  the  writer  (1887),  is 
52°  C.,  the  time  of  exposure  being  four  minutes  ;  a  few  colonies  only 
developed  after  exposure  to  a  temperature  of  50°  for  ten  minutes. 
In  Kitasato's  experiments  (1889)  ten  or  even  fifteen  minutes'  expo- 
sure to  a  temperature  of  55°  C.  was  not  always  successful  in  destroy- 
ing the  vitality  of  the  spirillum,  although  in  certain  cultures  exposure 
to  50°  for  fifteen  minutes  was  successful.  He  was  not,  however, 
able  to  find  any  difference  between  old  and  recent  cultures  as  regards 
resistance  to  heat  or  to  desiccation.  In  a  moist  condition  this  spiril- 
lum retains  its  vitality  for  months — as  much  as  nine  months  in  agar 
and  about  two  months  in  liquefied  gelatin.  It  is  quickly  destroyed 
by  desiccation,  as  first  determined  by  Koch,  who  found  that  it  did 
not  grow  after  two  or  three  hours  when  dried  in  a  thin  film  on  a 
glass  cover.  In  Kitasato's  experiments  (1889)  the  duration  of  vital- 
ity was  found  to  vary  from  a  few  hours  to  thirteen  days,  the  differ- 
ence depending  largely  upon  the  thickness  of  the  film.  When  dried 
upon  silk  threads  they  may  retain  their  vitality  for  a  considerably 
longer  time  (Kitasato).  Very  numerous  experiments  have  been 
made  to  determine  the  amount  of  various  disinfecting  agents  re- 
quired to  destroy  the  vitality  of  this  microorganism.  We  give  be- 
low the  results  recently  reported  by  Boer  (1890),  whose  experiments 
were  made  in  Koch's  laboratory.  Experiments  upon  a  culture  in 
bouillon  kept  for  twenty-four  hours  in  the  incubating  oven,  time  of 
exposure  two  hours  :  hydrochloric  acid,  1  : 1,350  ;  sulphuric  acid, 
1  : 1,300  ;  caustic  soda,  1  : 150  ;  ammonia,  1  :  350  ;  mercuric  cyanide, 
1  : 60,000  ;  gold  and  sodium  chloride,  1  : 1,000  ;  silver  nitrate,  1: 4,000; 
arsenite  of  soda,  1  : 400  ;  malachite  green,  1  :  5,000  ;  methyl  violet, 
1:1,000;  carbolic  acid,  1  : 400  ;  creolin,  1:3,000;  lysol,  1:500.  In 


598  PATHOGENIC    SPIRILLA. 

Bolton's  experiments  (1887)   mercuric  chloride  was  effective  in  two 
hours  in  the  proportion  of  1  : 10,000  ;  sulphate  of  copper,  1  : 500. 

The  low  thermal  death-point  and  comparatively  slight  resisting 
power  for  desiccation  and  chemical  agents  indicate  that  this  spiril- 
lum does  not  form  spores,  and  most  bacteriologists  agree  that  this 
is  the  case.  Hueppe,  however,  has  described  a  mode  of  spore  for- 
mation which  is  different  from  that  which  occurs  among  the  bacilli, 
viz.,  the  formation  of  so-called  arthrospores  ;  these  are  said  to  be 
developed  in  the  course  of  the  spiral  threads,  not  as  endogenous  re- 
fractive spores,  but  as  spherical  bodies  which  have  a  somewhat 
greater  diameter  than  the  filament  and  are  somewhat  more  refrac- 
tive. This  mode  of  spore  formation  has  not  been  observed  by  Kita- 
sato  and  other  bacteriologists  who  have  given  attention  to  the  ques- 
tion, and  cannot  be  considered  as  established.  In  competition  with 
the  ordinary  putrefactive  bacteria  the  cholera  spirillum  soon  disap- 
pears, and,  as  determined  by  Neffelman  and  by  Kitasato,  they  only 
survive  for  a  few  days  when  mixed  with  normal  faeces. 

A  test  for  the  presence  of  the  cholera  spirillum  has  been  found 
by  Bujwid  and  by  Dunham  in  the  reddish- violet  color  produced  in 
bouillon  cultures  containing  peptone,  or  in  cultures  in  nutrient  gela- 
tin, when  a  small  quantity  of  sulphuric  acid  is  added  to  the  culture. 
According  to  Frankel,  this  test  serves  to  distinguish  it  from  the  ordi- 
nary bacteria  of  the  intestine  and  from  the  Finkler-Prior  spirillum, 
but  not  from  Metschiiikoff's  spirillum  ("  vibrio").  The  reaction  is 
shown  by  bouillon  cultures  which  have  been  in  the  incubating  oven 
for  ten  or  twelve  hours,  and  by  gelatin  cultures  in  which  liquefac- 
tion has  occurred.  The  sulphuric  acid  used  should  be  quite  pure  ; 
the  color  quickly  appears  and  is  reddish- violet  or  purplish-red.  Ac- 
cording to  Salkowski,  the  red  color  is  due  to  the  well-known  indol 
reaction,  which  in  cultures  of  the  cholera  spirillum  is  exceptionally 
intense  and  rapid  in  its  development.  A  test  which  is  said  to  dis- 
tinguish cultures  of  the  cholera  spirillum  from  the  spirillum  of  De- 
neke  and  that  of  Finkler-Prior,  has  been  proposed  by  Cahen.  This 
consists  in  adding  a  solution  of  litmus  to  the  bouillon  and  in  making 
the  culture  at  37°  C.  The  cholera  cultures  show  on  the  following 
day  a  decoloration  which  does  not  occur  at  this  temperature  with  the 
other  spirilla  named. 

For  determining  as  promptly  as  possible  whether  certain  suspected 
excreta  contain  cholera  spirilla,  a  little  of  the  material  may  be  used 
to  inoculate  greatly  diluted  bouillon,  gelatin  plates  being  made  at 
the  same  time.  At  the  end  of  ten  or  twelve  hours  the  cholera  spiril- 
lum, if  present,  will  already  have  formed  a  characteristic  wrinkled 
film  upon  the  surface  ;  a  little  of  this  should  be  used  to  start  a  new 
culture  in  diluted  bouillon,  and  a  series  of  gelatin  plates  made  from 


PATHOGENIC    SPIRILLA. 


599 


it,  after  which  the  color  test  may  be  applied.  The  result  of  this,  in 
connection  with  the  morphology  of  the  microorganisms  forming  the 
film  and  the  character  of  growth  in  the  gelatin  plates,  will  estab- 
lish the  diagnosis  if  the  cholera  spirillum  is  present  in  considerable 
numbers.  If  but  few  are  present  in  the  original  material  it  may  be 
necessary  to  make  two  or  more  series  of  plates  and  bouillon  cultures 
before  a  pure  culture  can  be  obtained  and  a  positive  diagnosis  made. 


/• 


c  •* 


Fia.  179.— Section  through  mucous  membrane  of  intestine  from  cholera  cadaver;  a  tubular 
gland  (a)  is  cut  obliquely;  in  the  interior  of  this  (6),  and  between  the  epithelial  and  basement 
membrane,  are  numerous  spirilla.  X  600.  (Fliigge.) 

The  spirillum  is  not  found  in  the  blood  or  in  the  various  organs  of 
individuals  who  have  succumbed  to  an  attack  of  cholera,  but  it  is 
constantly  found  in  the  alvine  discharges  during  life  and  in  the  con- 
tents of  the  intestine  examined  immediately  after  death ;  frequently  in 
almost  a  pure  culture  in  the  colorless  "  rice-water"  discharges.  It  is 
evident,  therefore,  that  if  we  accept  it  as  the  etiological  agent  in  this 
disease,  the  morbid  phenomena  must  be  ascribed  to  the  absorption  of 
toxic  substances  formed  during  its  multiplication  in  the  intestine.  In 
cases  which  terminated  fatally  after  a  very  brief  sickness  Koch  found 
but  slight  changes  in  the  mucous  membrane  of  the  intestine,  which 
was  slightly  swollen  and  reddened ;  but  in  more  protracted  cases  the 
follicles  and  Peyer's  patches  were  reddened  around  their  margins,  and 
an  invasion  of  the  mucous  membrane  by  the  "  comma  bacilli "  was 
observed  in  properly  stained  sections ;  they  penetrated  especially 
the  follicles  of  Lieberkiihn,  and  in  some  cases  were  seen  between  the 
epithelium  and  basement  membrane.  As  a  rule,  the  spirillum  is  not 


600  PATHOGENIC   SPIRILLA. 

present  in  vomited  matters,  but  Koch  found  it  in  small  numbers  in 
two  cases  and  Nicati  and  Rietsch  in  three.  In  about  one  hundred 
cases  in  which  Koch  examined  the  excreta,  or  the  contents  of  the  in- 
testine of  recent  cadavers,  during  his  stay  in  Egypt,  in  India,  and  in 
Toulon,  his  "  comma  bacillus"  was  constantly  found,  and  other  ob- 
servers have  fully  confirmed  him  in  this  particular — Mcati  and 
Rietsch  in  thirty-one  cases  examined  at  Marseilles  ;  Pf eiff er,  twelve 
cases  in  Paris ;  Schottelius  in  cases  examined  in  Turin ;  Ceci  in 
Genoa,  etc.  On  the  other  hand,  very  numerous  control  experiments 
made  by  Koch  and  others  show  that  it  is  not  present  in  the  alvine 
discharges  of  healthy  persons  or  in  the  contents  of  the  intestine  of 
those  who  die  from  other  diseases.  In  the  writer's  extended  bacte- 
riological studies  of  the  excreta,  and  contents  of  the  intestine  of  ca- 
davers, in  yellow  fever,  he  has  not  once  encountered  any  microor- 
ganism resembling  the  cholera  spirillum. 

As  none  of  the  lower  animals  are  liable  to  contract  cholera  during 
the  prevalence  of  an  epidemic,  or  as  a  result  of  the  ingestion  of  food 
contaminated  with  choleraic  excreta,  we  have  no  reason  to  expect 
that  pure  cultures  of  the  spirillum  introduced  by  subcutaneous  inocu- 
lation or  by  the  mouth  will  give  rise  in  them  to  a  typical  attack  of 
cholera.  Moreover,  it  has  been  shown  by  experiment  that  this  spi- 
rillum is  very  sensitive  to  the  action  of  acids,  and  is  quickly  de- 
stroyed by  the  acid  secretions  of  the  stomach,  of  man  or  the  lower 
animals,  when  the  functions  of  this  organ  are  normally  performed. 
By  a  special  method  of  procedure,  however,  Nicati  and  Rietsch,  and 
Koch,  have  succeeded  in  producing  in  guinea-pigs  choleraic  symp- 
toms and  death.  The  first-named  investigators  injected  cultures  of 
the  spirillum  into  the  duodenum,  after  first  ligating  the  biliary  duct; 
the  animals  experimented  upon  died,  and  the  intestinal  contents  con- 
tained the  spirillum  in  large  numbers.  The  fact  that  this  procedure 
involves  a  serious  operation  which  alone  might  be  fatal,  detracts 
from  the  value  of  the  results  obtained.  Koch's  experiments  on 
guinea-pigs  are  more  satisfactory,  and,  having  been  fully  controlled 
by  comparative  experiments,  show  that  the  "  comma  bacillus "  is 
pathogenic  for  these  animals  when  introduced  in  a  living  condition 
into  the  intestine.  This  was  accomplished  by  first  neutralizing  the 
contents  of  the  stomach  with  a  solution  of  carbonate  of  soda — five 
cubic  centimetres  of  a  five-per-cent  solution,  injected  into  the  stomach 
through  a  pharyngeal  catheter.  For  the  purpose  of  restraining  in- 
testinal peristalsis  the  animal  also  receives,  in  the  cavity  of  the  abdo- 
men, a  tolerably  large  dose  of  laudanum — one  gramme  tincture  of 
opium  to  two  hundred  grammes  of  body  weight.  The  animals  are 
completely  narcotized  by  this  dose  for  about  half  an  hour,  but  re' 
cover  from  it  without  showing  any  ill  effects.  Soon  after  the  ad' 


PATHOGENIC    SPIRILLA.  601 

ministration  of  the  opium  a  bouillon  culture  of  the  cholera  spirillum 
is  injected  into  the  stomach  through  a  pharyngeal  catheter.  As  a 
result  of  this  procedure  the  animal  shows  an  indisposition  to  eat  and 
other  signs  of  sickness,  its  posterior  extremities  become  weak  and 
apparently  paralyzed,  and,  as  a  rule,  death  occurs  within  forty-eight 
hours.  At  the  autopsy  the  small  intestine  is  found  to  be  congested 
and  is  filled  with  a  watery  fluid  containing  the  spirillum  in  great 
numbers.  Comparatively  large  quantities  of  a  pure  culture  injected 
into  the  abdominal  cavity  of  rabbits  or  of  mice  often  produce  a  fatal 
result  within  two  or  three  hours  ;  and  Nicati  and  Rietsch  have  ob- 
tained experimental  evidence  of  the  pathogenic  power  of  filtered  cul- 
tures not  less  than  eight  days  old.  The  most  satisfactory  evidence 
that  this  spirillum  is  able  to  produce  cholera  in  man  is  afforded  by  an 
accidental  infection  which  occurred  in  Berlin  (1884),  in  the  case  of  a 
young  man  who  was  one  of  the  attendants  at  the  Imperial  Board  of 
Health  when  cholera  cultures  were  being  made  for  the  instruction  of 
students.  Through  some  neglect  the  spirillum  appears  to  have  been 
introduced  into  his  intestine,  for  he  suffered  a  typical  attack  of 
cholera,  attended  by  thirst,  frequent  watery  discharges,  cramps  in 
the  extremities,  and  partial  suppression  of  urine.  Fortunately  he 
recovered  ;  but  the  genuine  nature  of  the  attack  was  shown  by  the 
symptoms  and  by  the  abundant  presence  of  the  "  comma  bacillus" 
in  the  colorless,  watery  discharges  from  his  bowels.  Mcati  and 
Riet3eli  observed  a  certain  degree  of  attenuation  in  the  pathogenic 
power  of  the  spirillum  after  it  had  been  cultivated  for  a  considerable 
time  at  20°  to  25°  C.  ;  and  the  observation  has  since  been  made  that 
cultures  which  have  been  kept  up  from  Koch's  original  stock  have 
no  longer  the  primitive  pathogenic  potency. 

Cunningham,  as  a  result  of  researches  made  in  Calcutta  (1891), 
arrives  at  the  conclusion  that  Koch's  "comma  bacillus"  cannot 
be  accepted  as  the  specific  etiological  agent  in  this  disease.  This 
conclusion  is  based  upon  the  results  of  his  own  bacteriological 
studies,  which  may  be  summed  up  as  follows  :  First,  in  many  un- 
doubted cases  of  cholera  he  has  failed  to  find  comma  bacilli.  Sec- 
ond, in  one  case  he  found  three  different  species.  Third,  in  one  case 
the  reaction  with  acids  could  not  be  obtained.  From  sixteen  cases 
in  which  Cunningham  made  cultures  he  obtained  ten  different  vari- 
eties of  comma  bacilli,  the  characters  of  which  he  gives  in  his  pub- 
lished report.  It  may  be  that  in  India,  which  appears  to  be  the 
permanent  habitat  of  the  cholera  spirillum,  many  varieties  of  this 
microorganism  exist ;  but  extended  researches  made  in  the  laborato- 
ries of  Europe  show  that  Cunningham  is  mistaken  in  supposing  that 
spirilla  resembling  Koch's  "  comma  bacillus  "  are  commonly  present 
in  the  intestine  of  healthy  persons.  The  view  advocated  is  that 


G02 


PATHOGENIC   SPIRILLA. 


during  the  attack  these  spirilla  are  found  in  increased  numbers  be- 
cause conditions  are  more  favorable  for  their  development,  but  that 
they  have  no  etiological  import.  The  writer  would  remark  that,  in 
very  extended  researches  made  in  the  United  States  and  in  Cuba,  he 
has  never  found  any  microorganism  resembling  Koch's  cholera  spi- 
rillum in  the  faeces  of  patients  with  yellow  fever  or  of  healthy  indi- 
viduals, or  in  the  intestinal  contents  of  yellow-fever  cadavers. 

SPIRILLUM   OF   FINKLER   AND    PRIOR. 

Synonym. — Vibrio  proteus.  - 

Obtained  by  Finkler  and  Prior  (1884)  from  the  faeces  of  patients   with 
cholera  nostras,  after  allowing  the  dejecta  to  stand  for  some  days.     Subse- 


Fio.  180. 


FIG.  181. 


FIG.  182. 

X  1,000.   From  a  photomicro- 


FIG.  180.— Spirillum  of  Finkler  and  Prior,  from  a  gelatin  culture, 
graph.    (Frankel  and  Pfeiffer.) 

Fia.  181.— Spirillum  of  Finkler  an!  Prior;  colonies  upon  gelatin  plate;  a,  end  of  sixteen  hours; 
6,  end  of  twenty-four  hours;  c,  end  of  thirty-six  hours,  x  80.  (Fliigge  ) 

FIG.  182.— Spirillum  of  Finkler  and  Prior;  culture  in  nutrient  gelatin;  c,  two  days  old;  d,  four 
days  old.  (Flugge.) 

quent  researches  have  not  sustained  the  view  that  this  spirillum  is  the  speci- 
fic cause  of  cholera  morbus. 

Morphology. — Resembles  the  spirillum  of  Asiatic  cholera,  but  the  curved 
segments  ("  bacilli")  are  somewhat  longer  and  thicker  and  not  so  uniform 
in  diameter,  the  central  portion  being  usually  thicker  than  the  somewhat 
pointed  ends;  forms  spiral  filaments,  which  are  not  as  numerous,  and  are 
usually  shorter  than  those  formed  by  the  cholera  spirillum.  In  unfavorable 
media  involution  forms  are  common — large  oval,  spherical,  or  spindle- 
shaped  cells,  etc.  Has  a  single  flagellum  at  one  end  of  the  curved  segments, 
which  is  from  one  to  one  and  one-half  times  as  long  as  these. 

Stains  with  the  usual  aniline  colors — best  with  an  aqueous  solution  of 
fucbsin. 


PATHOGENIC    SPIRILLA. 

Biological  Characters. — An  aerobic  and  facultative  anaerobic,  liquefy- 
ing, motile  spirillum.  Spore  formation  not  demonstrated.  Grows  in  the 
usual  culture  media  at  the  room  temperature.  Upon  gelatin  plates  small, 
white,  punctiform  colonies  are  developed  at  the  end  of  twenty  four  hours, 
which  under  the  microscope  are  seen  to  be  finely  granular  and  yellowish  or 
yellowish-brown  in  color ;  liquefaction  of  the  gelatin  around  these  colonies 
progresses  rapidly,  and  at  the  end  of  forty-eight  hours  is  usually  complete  in 
plates  where  they  are  numerous.  Isolated  colonies  on  the  second  day  form 
saucer-shaped  depressions  in  the  gelatin  the  size  of  lentils,  having  a  sharply 
denned  border.  In  gelatin  stab  cultures  liquefaction  progresses  much  more 
rapidly  than  in  similar  cultures  of  the  cholera  spirillum,  and  a  stocking- 
shaped  pouch  of  liquefied  gelatin  is  already  seen  on  the  second  day,  which 
rapidly  increases  in  dimensions,  so  that  by  the  end  of  a  week  the  gelatin  is 
usually  completely  liquefied ;  upon  the  surface  of  the  liquefied  medium  a 
whitish  film  is  seen.  Upon  agar  a  moist,  slimy  layer,  covering  the  entire 
surface,  is  quickly  developed.  The  growth  in  blood  serum  is  rapid  and 
causes  liquefaction  of  the  medium.  \Jponpotato  this  spirillum  grows  at  the 
room  temperature  and  produces  a  slimy,  grayish-yellow,  glistening  layer, 
which  soon  extends  over  the  entire  surface.  The  cholera  spirillum  does  not 
grow  upon  potato  at  the  room  temperature.  The  cultures  of  the  Finkler- 
.  Prior  spirillum  give  off  a  tolerably  strong  putrefactive  odor,  and,  according 
to  Buchner,  in  media  containing  sujrar  an  acid  reaction  is  produced  as  a  re- 
sult of  their  development.  Th«y  have  a  greater  resistance  to  desiccation  than 
the  cholera  spirillum. 

Pathogenesis. — Pathogenic  for  guinea-pigs  when  injected  into  the 
stomach  by  Koch's  method,  after  previous  injection  of  a  solution  of  car- 
bonate of  soda,  but  a  smaller  proportion  of  the  animals  die  from  such  injec- 
tions (Koch).  At  the  autopsy  the  intestine  is  pale,  and  its  watery  contents, 
which  contain  the  spirilla  in  great  numbers,  have  a  penetrating,  putrefactive 
odor. 

SPIRILLUM   TYROGENUM. 

Synonyms  — Spirillum  of  Deneke;  Kasespirillen. 

Obtained  by  Deneke  (1885)  from  old  cheese. 

Morphology. — Curved  rods  and  long,  spiral  filaments  resembling   the 
spirilla  of  Asiatic  cholera.     The  diameter  of  the  curved  segments  is  some- 
what less  than  that  of  the  cholera  spirillum,  and  the  turns  in  the  spiral  fila- 
ments are  lower  and  closer  together.    The  diame- 
ter of  the  "commas"  is  uniform  throughout,  so 
that  this  spirillum  more  closely  resembles  the 
cholera  spirillum  than  does  that  of  Finkler  and 
Prior. 

Stains  with  the  usual   aniline    colors — best 
with  an  aqueous  solution  of  fuchsin. 

Biological  Characters. — An  aerobic  and  fac- 
ultative anaerobic,  liquefying,  motile  spirillum.  ^^ 
Spore  formation  not    demonstrated.     Grows  in 

the  usual  culture  media  at  the  room  temperature        FIG.   183.— Spirillum  tyroge- 
— more  rapidly  than  the  cholera  spirillum  and     num.    x  TOO.   (Flugge.) 
less  so  than  that  of  Finkler  and  Prior.      Upon 

gelatin  plates  small,  punctiform  colonies  are  developed,  which  on  the  second 
day  are  about  the  size  of  a  pin's  head  and  have  a  yellowish  color;  under 
the  microscope  they  are  seen  to  be  coarsely  granular,  of  a  yellowish-green 
color  in  the  centre  and  paler  towards  the  margins.  The  outlines  of  the  colo- 
nies are  sharply  defined  at  first,  but  later,  when  liquefaction  has  commenced, 
the  sharp  contour  is  no  longer  seen.  At  first  liquefaction  of  the  gelatin 
causes  funnel-shaped  cavities  resembling  those  formed  by  the  cholera  spiril- 
lum, but  liquefaction  is  more  rapid.  In  gelatin  stab  cultures  liquefaction 
occurs  all  along  the  line  of  puncture,  and  the  spirilla  sink  to  the  bottom  of 


004 


PATHOGENIC    SPIRILLA. 


the  liquefied  gelatin  in  the  form  of  a  coiled  mass,  while  a  thin,  yeflowish 
layer  forms  upon  the  surface ;  complete  liquefaction  usually  occurs  in 
about  two  weeks.  Upon  the  surface  of  agar  a  thin,  yellowish  layer  forms 


FIG.  184.— Spirillum    tyrogenum;  colonies  in    gelatin  plate;  a,  end   of  sixteen  hours;   7>,   end  of 
twenty-four  hours ;  c,  end  of  thirty-six  hours.    X  80.    (Fliigge.) 

along  the  impfstrich.  Upon  potato,  at  a  temperature  of  37°  C.,  a  thin,  yel- 
low layer  is  usually  developed  (not  always — Eisenberg) ;  this  contains,  as  a 
rule,  beautifully  formed,  long1,  spiral  filaments. 

Pathogenesis. — Pathogenic  for  guinea-pigs  when  introduced  into  the 
stomach  by  Koch's  method  ;  three  out  of  fifteen  animals  treated  in  this  way 
succumbed. 


SPIRILLUM   METSCHNIKOVI. 

Synonym. — Vibrio  Metschnikovi  (Gameleia). 

Obtained  by  Gamele'ia  (1888)  from  the  intestinal  contents  of  chickens 
dying  of  an  infectious  disease  which  prevails  in  certain  parts  of  Russia  dur- 
ing the  summer  months,  and  which  in  some  respects  re- 
sembles fowl  cholera.  The  experiments  of  Gamele'ia  show 
that  the  spirillum  under  consideration  is  the  cause  of  the 
disease  referred  to,  which  he  calls  gastro-enteritischol erica. 
Morphology.— Curved  rods  with  rounded  ends,  and  spi- 
ral filaments  ;  the  curved  segments  are  usually  somewhat 
shorter,  thicker,  and  more  decidedly  curved  than  the 
"  comma  bacillus"  of  Koch.  The  size  differs  very  consid- 
erably in  the  blood  of  inoculated  pigeons,  the  diametei*- 
being  sometimes  twice  as  great  as  that  of  the  cholera  spiril- 
lum, and  at  others  about  the  same.  A  single,  long,  undu- 
lating flagellum  may  be  seen  at  one  extremity  of  the  spiral 
filaments  or  curved  rods  in  properly  stained  preparations. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's 
method. 

Biological  Characters. — An  aerobic  (facultative  an- 
aerobic ?),  liquefying,  ni(/tile  spirillum.  According  to 
Gamale'ia,  endogenous  spores  are  formed  by  this  spirillum  ; 
but  Pfeiffer  does  not  confirm  this  observation,  and  it  must 
be  considered  extremely  doubtful  in  view  of  the  slight 
resistance  to  heat — killed  in  five  minutes  by  a  temperature 
of  50°  C.  Grows  in  the  usual  culture  media  at  the  room 
temperature.  Upon  gelatin  plates  small,  white,  puncti- 
form  colonies  are  developed  at  the  end  of  twelve  to  six- 
teen hours ;  these  rapidly  increase  in  size  and  cause  lique- 
faction of  the  gelatin,  which  is,  however,  much  more  rapid 
with  some  than  with  others.  At  the  end  of  three  days 
large,  saucer-like  areas  of  liquefaction  may  be  seen  resem- 
bling that  produced  by  the  Finkler-Prior  spirillum  and  the 
contents  of  which  are  turbid,  while  other  colonies  have 
produced  small,  funnel-shaped  cavities  filled  with  transparent,  liquefied  gel- 
atin and  resembling  colonies  of  the  cholera  spirillum  of  th?  *snne  age.  Under 


FIG.  185.-Spiril- 
lum  Metschnikovi ; 
culture  in  nutrient 
gelatin,  end  of  forty- 
eight  hours.  From  a 
photograph.  (Fran- 
kel  and  Pfeiffer.) 


PATHOGENIC    SPIRILLA.  605 

the  microscope  the  larger  liquefied  areas  are  seen  to  contain  yellowish-brown 
granular  masses  which  are  in  active  movement,  and  the  margins  are  sur- 
rounded by  a  border  of  radiating  filaments.  In  gelatin  stab  cultures  the 
growth  resembles  that  of  the  cholera  spirillum,  but  the  development  is  more 
rapid.  Upon  agar,  at  37°  C.,  a  yellowish  layer  resembling  that  formed  by 
the  cholera  spirillum  is  quickly  developed.  Upon  potato  no  growth  occurs 
at  the  room  temperature,  but  at  37°  C.  a  yellowish-brown  or  chocolate-col- 
ored layer  is  formed,  which  closely  resembles  that  produced  by  the  cholera 
spirillum  under  the  same  circumstances.  In  bouillon,  at  37°  C.,  develop- 
ment is  extremely  rapid,  and  the  liquid  becomes  clouded  and  opaque,  having 
a  grayish-white  color,  while  a  thin,  wrinkled  film  forms  upon  the  surface. 
When  muriatic  or  sulphuric  acid  is  added  to  a  culture  in  peptomzed  bouillon 
a  red  color  is  produced  similar  to  that  produced  in  cultures  of  the  cholera 
spirillum,  and  even  more  pronounced.  In  milk,  at  35°  C.,  rapid  development 
occurs,  and  the  milk  is  coagulated  at  the  end  of  a  week ;  the  precipitated 
casein  accumulates  at  the  bottom  of  the  tube  in  irregular  masses  and  is  not 
redissolved.  The  milk  acquires  a  strongly  acid  reaction  and  the  spirilla 
quickly  perish. 

Pathogenesis. — Pathogenic  for  chickens,  pigeons,  and  guinea-pigs;  rab- 
bits and  mice  are  refractory  except  for  very  large  doses.  Chickens  suffering 
from  the  infectious  disease  caused  by  this  spirillum  remain  quiet  and  somno- 
lent, with  ruffled  feathers ;  they  have  diarrhoea ;  the  temperature  is  not  ele- 
vated above  the  normal,  as  is  the  case  in  chicken  cholera.  At  the  autopsy 
the  most  constant  appearance  is  hypersemia  of  the  entire  alimentary  canal. 
A  grayish-yellow  liquid,  more  or  less  mixed  with  blood,  is  found  in  con- 
siderable quantity  in  the  small  intestine ;  the  spleen  is  not  enlarged  and  the 
organs  generally  are  normal  in  appearance.  In  adult  chickens  the  spirillum 
is  not  found  in  the  blood,  but  in  young  ones  its  presence  may  be  verified  by 
the  culture  method  and  by  inoculation  into  pigeons,  which  die  in  from 
twelve  to  twenty  hours  after  being  inoculated  with  two  to  four  cubic  cen- 
timetres. The  pathological  appearances  in  pigeons  correspond  with  those 
found  in  chickens,  but  usually  the  spirillum  is  found  in  great  numbers  in 
blood  taken  from  the  heart.  A  few  drops  of  a  pure  culture  inoculated  sub- 
cutaneously  in  pigeons  or  injected  into  the  muscles  cause  their  death  in 
eight  to  twelve  hours.  Gameleia  claims  that  the  virulence  of  cultures  is 
greatly  increased  by  successive  inoculations  in  pigeons,  but  Pfeiffer  has 
shown  that  very  minute  doses  are  fatal  to  pigeons  and  that  no  decided  in- 
crease of  virulence  occurs  as  a  result  of  successive  inoculations.  According 
to  Gameleia,  chickens  may  be  infected  by  giving  them  food  contaminated 
with  the  cultures  of  the  spirillum,  but  pigeons  resist  infection  in  this  way. 
Guinea-pigs  usually  die  in  from  twenty  to  twenty-four  hours  after  receiving 
a  subcutaneous  inoculation  ;  at  the  autopsy  an  extensive  subcutaneous 
oedema  is  found  in  the  vicinity  of  the  point  of  inoculation,  and  a  superficial 
necrosis  may  be  observed  ;  the  blood  and  the  organs  generally  contain  the 
"  vibrio  "  in  great  numbers,  showing  that  the  animals  die  from  general  in- 
fection— acute  septicaemia.  When  infection  occurs  in  these  animals  by  way 
of  the  stomach  the  intestine  will  be  found  highly  inflamed  and  its  liquid  con- 
tents will  contain  numerous  spirilla. 

Gameleia  has  shown  that  pigeons  and  guinea-pigs  may  be  made  immune 
by  inoculating  them  with  sterilized  cultures  of  the  spirillum— sterilized  by 
heat  at  100°  C.  Old  cultures  contain  more  of  the  toxic  substance  than  those 
of  recent  date.  Thus  two  to  three  cubic  centimetres  of  a  culture  twenty  days 
old  will  kill  a  guinea-pig  when  injected  subcutaneously,  while  five  cubic 
centimetres  of  a  culture  five  days  old  usually  fail  to  do  so.  According  to 
Pfeiffer,  old  cultures  have  a  decidedly  alkaline  reaction,  and  their  toxic  power 
is  neutralized  by  the  addition  of  sulphuric  acid. 

Gameleia  has  claimed  that  by  passing  the  cholera  spirillum  of  Koch 
through  a  series  of  pigeons,  by  successive  inoculation,  its  pathogenic  power 


606  PATHOGENIC   SPIRILLA. 

is  greatly  increased,  and  that  when  sterilized  cultures  of  this  virulent  vari- 
ety of  the  "  comma  bacillus  "  are  injected  into  pigeons  they  become  immune 
against  the  pathogenic  action  of  the  "  vibrio  Metschnikoff , "  and  the  reverse. 
Pfeiffer  (1889),  in  an  extended  and  carefully  conducted  research,  was  not 
able  to  obtain  any  evidence  in  support  of  this  claim. 

NOTES  RELATING  TO  THE  PATHOGENIC  SPIRILLA. 

Quite  a  number  of  spirilla  have  been  obtained  from  various  sources 
which  resemble  more  or  less  closely  the  spirillum  of  Asiatic  cholera. 
It  appears  probable  that  some  of  these  are  in  fact  varieties  of  Koch's 
"  comma  bacillus  "  which  have  undergone  various  modifications  as  a 
result  of  the  conditions  under  which  they  have  maintained  their  ex- 
istence as  saprophytes.  Others  are  evidently  essentially  different, 
and  have  no  very  near  relationship  to  the  cholera  spirillum.  The 
principal  points  of  difference  between  these  recently  described  spirilla 
and  Spirillum  cholerse  Asiatics  are  given  in  the  following  resume, 
for  which  we  are  indebted  to  Dieudonne  (1804). 

' '  Since  the  outbreak  of  cholera  in  1892,  various  vibrios  have  been  de- 
scribed which  resemble  more  or  less  closely  the  cholera  vibrio.  When  these 
are  tested  as  to  their  morphological  characters,  growth  in  peptone  solutions, 
in  gelatin  and  agar  plates,  cholera-red  reaction,  and  pathogenic  power,  they 
may  be  divided,  at  the  outset,  into  two  groups  :  viz.,  such  vibrios  as  show 
only  a  remote  resemblance  to  the  cholera  vibrio,  and  therefore  are  easily  dif- 
ferentiated from  it,  and  such  as  present  only  minor  differences  or  none  at 
all  that  have  been  demonstrated. 

"To  the  first  group  belongs  the  spirillum  isolated  by  Russell  from  sea 
water — Spirillum  marinum — which  rapidly  liquefies  gelatin  and  does  not 
grow  at  the  body  temperature.  Reiion  isolated  from  water,  obtained  at  Bil- 
lancourt,  a  vibrio  which  likewise  quickly  liquefies  gelatin,  but  is  not  patho- 
genic for  guinea-pigs,  either  by  subcutaneous  or  intraperitoneal  inoculation. 
Grunther,  in  examining  the  Spree  water,  found  a  vibrio  which,  upon  gelatin 
plates,  formed  circular  colonies  with  smooth  margins,  very  finely  granular 
and  of  a  brown  color.  This  vibrio  did  not  give  the  indol  reaction,  and  all 
infection  experiments  gave  a  negative  result.  Giinther  named  this  sapro- 
phyte Vibrio  aquatilis.  About  the  same  time  (1892)  Kiessling  obtained  from 
water,  from  Blankenese,  a  vibrio  which  presented  similar  characters  and 
probably  is  identical  with  that  of  Giinther.  Weibel  obtained  from  well-water 
a  vibrio  which  liquefies  gelatin  more  rapidly  than  the  cholera  vibrio  ;  its 
pathogenic  action  was  not  tested.  Bujwid  (1893)  isolated  from  Weichsel 
water  a  vibrio  which  at  low  temperatures  (12°  C.)  grew  almost  the  same  as 
the  cholera  vibrio,  but  at  higher  temperatures  was  easily  distinguished  from 
it.  Bujwid's  assistant,  Orlowski,  found  in  a  well  at  Lubin  a  very  similar 
vibrio.  Loffler  (1893)  obtained  from  the  Peeiie  water  a  vibrio  which  at  37° 
C.  grows  rapidly  and  liquefies  gelatin  very  rapidly,  like  the  Finkler-Prior 
spirillum.  Fokker  (1893),  from  water  of  the  harbor  at  Groiiingeii,  obtained 
a  vibrio  which  rapidly  liquefied  gelatin  and  occasionally  gave  the  indol  re- 
action. Injections  into  the  peritoneal  cavity  of  mice  and  guinea-pigs  gave 
a  negative  result.  Fokker  supposes  that  this  is  an  attenuated  cholera  bacil- 
lus, because  it  forms  the  same  ensyme  as  cholera  bacteria,  and  when  culti- 
vated for  three  months  its  characters,  especially  its  peptonizing  power,  had 
changed.  Fischer  (1893)  found  in  the  stools  of  a  woman  suffering  from  diar- 
rhoea a  vibrio  which  in  gelatin  cultures  resembled  that  of  Finkler  and 
Prior.  In  bouillon  and  peptone  solution  it  caused  clouding  and  formation  of 


PATHOGENIC   SPIRILLA.  607 

a  pellicle,  but  only  gave  a  slight  indol  reaction.  A  portion  of  the  mice  in- 
oculated subcutaiieously  had  after  a  time  abscesses,  from  the  contents  of 
which  Fischer  was  able  to  cultivate  his  vibrio,  which  he  named  Vibrio  helco- 
genes.  Vogler  (1893),  in  an  extended  series  of  examinations  of  faeces,  found 
a  vibrio  which  showed  manv  points  of  resemblance  to  the  cholera  vibrio  in 
its  growth  in  gelatin.  But  it  constantly  gave  a  negative  indol  reaction,  and 
was  not  pathogenic  for  guinea-pigs  when  injected  into  the  peritoneal  cavity. 
Bleisch  obtained  from  the  dejecta  of  a  man  who  died  with  choleraic  symptoms 
a  bacterium  which  upon  gelatin  plates  grew  at  first  like  the  cholera  bacillus,  but 
was  distinguished  from  it  by  many  points  of  difference  in  other  respects : 
short  rods,  sometimes  bent,  but  never  showing  spiral  forms.  It  gave  the 
cholera-red  reaction.  Wolf  (1883)  obtained  from  cervical  secretion,  from  a 
woman  suffering  from  chronic  eiidometritis,  a  comma-formed  bacillus,  which 
in  its  growth  011  gelatin  plates  resembled  the  cholera  vibrio.  The  liquefac- 
tion was,  however,  much  more  rapid,  a  culture  a  day  old  being  as  far  ad- 
vanced as  a  cholera  culture  of  three  to  four  days.  The  addition  of  sulphuric 
acid  to  a  bouillon  culture  caused  a  faint  rose-red  color,  which  upon  standing 
changed  to  brown.  The  addition  of  sulphuric  acid  and  potassium  iodide  paste 
did  not  cause  a  blue  color,  so  there  was  110  formation  of  nitrites.  Bonhoff 
(1893),  in  water  from  Stolpe,  in  Pommerania,  discovered  two  vibrios,  one  of 
which  in  the  first  twenty-four  hours  grevsr  like  the  cholera  vibrio,  but  did  not 
give  the  cholera-red  reaction.  Out  of  four  guinea-pigs  inoculated  one  only 
died  with  cholera-like  symptoms.  The  other  vibrio  gave  the  cholera-red  reac- 
tion, but  did  not  liquefy  gelatin  and  was  very  inconstant  as  regards  its  patho- 
genic power.  Zorkendorfer  (1893)  isolated  a  vibrio  from  the  stools  of  a 
woman  who  died  with  choleraic  symptoms,  which  at  first  grew  upon  gelatin 
plates  like  the  cholera  vibrio,  but  after  the  second  day  liquefied  the  gelatin 
very  rapidly,  so  that  it  could  no  longer  be  taken  for  the  same.  The  indol 
reaction  was  constantly  absent,  and  it  was  not  pathogenic  for  guinea-pigs, 
rabbits,  or  pigeons.  Blackstein  (1893)  obtained  from  the  water  of  the  Seine 
a  comma  bacillus  which  resembled  the  cholera  vibrio  in  many  particulars,  but 
was  distinguished  by  the  finer  granulation  and  more  opaque  appearance  of 
its  colonies.  Sanarelli  (1893),  by  the  use  of  special  media,  isolated  from  the 
water  of  the  Seine  and  of  the  Marne  110  less  than  thirty-two  vibrios,  four  of 
which  resembled  the  cholera  vibrio  in  giving  the  indol  reaction.  Three 
others  gave  the  indol  reaction  after  eight  days  ;  the  remainder  did  not  give  it 
at  ail,  or  only  very  faintly.  The  vibrios  which  upon  a  first  inoculation  gave 
110  results  or  only  very  slight  evidence  of  pathogenic  power,  when  carried 
through  a  series  of  animals  caused  a  fatal  infection.  When  a  sterilized  cul- 
ture of  the  colon  bacillus  was  injected  at  the  same  time  death  always  oc- 
curred. Sanarelli  believes  that  these  vibrios  must  have  had  a  common  ori- 
gin— from  the  dejecta  of  cholera  patients.  Fischer  (1894)  has  described  a 
number  of  vibrios  from  sea-water  which  are  distinguished  from  the  cholera 
vibrio  especially  by  a  preference  for  media  containing  sea-water.  Finally, 
the  vibrios  found  in  water,  referred  to  by  Koch  ('  Ueber  den  augenblicklicheii 
Stand  der  Cholera-diagnose,'  Zeitschr.  fur  Hygiene,  Bd.  xiv.,  page  319), 
belong  here. 

"Quite  different  from  these  is  a  second  group  of  vibrios  which  in  their  in- 
vestigation offered  great  and  often  almost  insuperable  difficulties  for  the 
differential  diagnosis.  Here,  first  of  all,  is  the  Vibrio  Berolinensis,  found  by 
Neisser  in  August,  1893,  and  described  by  Rubner,  Neisser,  and  Giinther. 
This  was  isolated  from  water  which  had  previously  contained  cholera  vibrios, 
for  which  reason  Dun  bar  considers  it  not  impossible  that  this  is  a  genuine 
cholera  vibrio,  somewhat  changed  perhaps  by  long-continued  development 
in  water.  Neither  in  its  morphology  nor  in  its  behavior  in  gelatin  stick  cul- 
tures, in  milk  and  other  media,  could  it  be  distinguished  from  the  genuine 
comma  bacillus  ;  the  indol  reaction  and  pathogenic  action  upon  guinea-pigs 
were  the  same  ;  on  the  contrary,  a  differentiation  was  easily  made  in  gelatin 
plate  cultures.  At  the  end  of  twenty -four  hours  it  formed  small,  spherical, 


G08  PATHOGENIC   SPIRILLA. 

finely  granular  colonies,  which  at  the  end  of  forty-eight  hours  were  not  yet 
visible  to  the  naked  eye.  Heider  (1893)  isolated  from  the  water  of  the  Donau 
canal  a  vibrio  which  he  called  Vibrio  Danubicus.  This  resembles  the  chol- 
era vibrio  fully  in  its  morphology.  As  a  distinguishing  character  it  was 
found  that  this  vibrio,  in  thinly  planted  plates,  forms  flat,  superficial  colo- 
nies having  irregularly  rounded  margins  and  other  slight  differences ;  also 
the  pathogenic  action  upon  mice  inoculated  subcutaneously,  and  the  ease  with 
which  guinea-pigs  are  infected  by  way  of  the  respiratory  passages.  It  is 
worthy  of  note  that  the  day  after  the  sample  was  taken  a  man  was  taken  sick 
with  cholera  who  had  worked  on  the  Donau  the  day  before — on  the  principal 
stream  at  a  place  far  below  the  junction  of  the  canal.  Dunbar  (1893)  found 
vibrios  in  the  Elbe,  in  the  Rhine,  in  the  Pegnitz,  and  in  the  Amstel  at  Amster- 
dam. These  presented  no  decided  characters  by  which  he  was  able  to  differ- 
entiate them  from  the  cholera  vibrio.  The  most  careful  comparative  investi- 
gations did  not  lead  to  the  discovery  of  any  points  of  difference  which  had 
not  already  been  observed  in  genuine  cholera  cultures.  Everything,  there- 
fore, indicated  that  these  were  genuine  cholera  bacilli,  especially  as  these 
vibrios  disappeared  from  the  rivers  when  cholera  ceased  to  prevail.  It  was 
first  possible  through  an  observation  of  Kutscher's  to  differentiate  a  portion 
of  these  water  bacteria,  and  certain  vibrios  isolated  from  the  discharges  of 
persons  suspected  of  having  cholera  from  cultures  of  the  cholera  spirillum. 
In  the  presence  of  oxygen,  at  a  suitable  temperature,  they  give  off  a  greenish- 
white  phosphorescence. 

"As  phosphorescence  has  never  been  observed  in  undoubted  cholera  cul- 
tures, we  can  assert  with  tolerable  certainty  that  such  phosphorescent  vibrios 
are  not  genuine  cholera  bacteria.  But  as  this  phosphorescent  property  was 
inconstant  in  thirty-eight  out  of  sixty-eight  cultures,  Dunbar  believes  that 
some  reserve  must  be  exercised  in  accepting  this  as  evidence  that  these  are 
not  genuine  cholera  vibrios.  Maasseii  (1894)  gives  as  a  further  distinguishing 
character  of  these  phosphorescent  vibrios  the  fact  that  they  form  a  strong, 
usually  wrinkled  pellicle  in  bouillon,  of  proper  alkalinity,  containing  gly- 
cerin or  carbohydrates  (cane  sugar,  lactose) ;  also  that  in  such  media  the 
formation  of  indol  and  a  subsequent  return  to  an  alkaline  reaction  may  be 
observed. 

"As  already  stated,  Sanarelli  isolated  from  Seine  water  a  considerable 
number  of  vibrios,  and  among  them  four — viz.  :  one  from  St.  Cloud,  Point- 
du-Jour,  Gennevilliers  No.  5,  and  Versailles  (Seine),  which  after  twenty-four 
hours  gave  a  distinct  indol  reaction  and  were  more  or  less  pathogenic  for 
guinea-pigs  (the  one  from  St.  Cloud  was  also  pathogenic  for  pigeons}.  I  van- 
off  (1893)  describes  a  vibrio  which  he  isolated  from  the  faeces  of  a  patient  with 
typhoid  fever.  But  as  the  discharges  had  been  mixed  with  Berlin  hydrant 
water,  Ivanoff  admits  the  possibility  that  his  vibrio  came  from  this  water. 
It  closely  resembles  the  cholera  vibrio,  but  is  distinguished  by  its  colonies  in 
gelatin  plates,  which,  at  the  end  of  twenty -four  to  thirty-six  hours,  in  place 
of  the  usual  coarse  granulation  of  cholera  colonies  shows  a  distinct  formation 
of  filaments.  Morphologically  the  vibrio  is  distinguished  by  a  decided  ten- 
dency to  preserve  the  spiral  form,  and  especially  by  its  size.  Celli  and  Saii- 
tori  (1893)  describe  a  Vibrio  romanus,  which  they  isolated  from  twelve 
undoubted  cases  of  cholera.  This  does  not  give  the  indol  reaction,  is  not 
pathogenic  for  animals,  and  does  not  grow  in  bouillon  or  agar  at  37°  C. 
This  is  considered  by  the  authors  named  an  atypical  variety  of  the  cholera 
vibrio,  especially  as  the  distinguishing  characters  did  not  prove  to  be  perma- 
nent. After  eight  months'  cultivation  the  cultures  gave  the  indol  reaction,  but 
the  pathogenic  power  was  still  almost  absent.  Recently  Chaiitemesse  (1894) 
has  described  a  vibrio  which  he  found  in  the  spring  of  1894  during  the  chol- 
era epidemic  at  Lisbon.  This  differed  in  many  particulars  from  the  genuine 
cholera  vibrio,  resembling  more  closely  the  vibrio  of  Finkler-Prior.  As  in 
the  Lisbon  epidemic,  with  a  large  number  taken  sick,  only  one  death  occurred, 
and  in  view  of  the  results  of  the  bacteriological  examination,  Chantemesse 


PATHOGENIC    SPIRILLA.  609 

supposes  this  to  have  been  an  epidemic  of  cholera  nostras.  Finally,  Pfuhl 
(1894)  found  a  vibrio  in  the  north  harbor  of  Berlin  which  from  its  growth  in 
gelatin  and  pathogenesis  for  pigeons  he  believes  to  be  identical  with  Vibrio 
Metschnikovi." 

To  the  list  of  vibrios  above  referred  to  as  resembling  more  or  less 
closely  the  cholera  spirillum  we  must  add  those  described  by  Cun- 
ningham (1894)  and  obtained  by  him  from  the  discharges  of  cholera 
patients.  He  has  described  "thirteen  distinct  forms  obtained  from 
cases  of  cholera  and  one  of  non-choleraic  origin." 

Pfeiffer  and  Issaeff  (1894)  report  that  they  have  found  a  sen- 
sitive test  for  the  differentiation  of  these  vibrios  in  the  specific 
character  of  cholera  immunity.  They  found  that  guinea-pigs  which 
were  immunized  against  cholera  infection  have  a  lasting  immunity, 
and  that  the  serum  of  such  immunized  animals  has  a  specific  ac- 
tion in  protecting  against  infection  by  genuine  cholera  vibrios 
only,  while  for  other  species  it  has  no  action  different  from  that 
of  the  blood  serum  of  normal  animals.  In  all  cases  where  the  cholera 
serum  acted  specifically  the  vibrios  were  promptly  destroyed,  while 
in  cases  where  this  specific  action  was  absent  the  injected  vibrios 
multiplied  rapidly  and  caused  the  death  of  the  animal.  By  means  of 
this  method  the  vibrios  isolated  from  water — the  phosphorescent 
vibrios  of  D unbar,  Vibrio  Danubicus,  Cholera  Massanah — are  shtfwn 
to  be  distinct  species,  while  the  vibrio  of  Ivanoff  behaves  like  the 
genuine  cholera  vibrio.  In  a  subsequent  paper  Pfeiffer  reports  the 
interesting  fact  that  a  trace  of  highly  active  cholera  serum,  added  to 
a  culture  of  the  cholera  spirillum,  when  injected  into  the  peritoneal 
cavity  of  a  guinea-pig,  within  a  surprisingly  brief  time  causes  the 
destruction  of  the  cholera  vibrios ;  whereas  no  such  effect  is  produced 
upon  other  species.  A  similar  destruction  occurs  when  cholera  vib- 
rios are  injected  into  the  abdominal  cavity  of  immunized  guinea- 
pigs.  The  researches  of  Dunbar  (1894)  indicate  that  Pfeiffer 's  test 
is  not  so  reliable  as  he  supposed ;  and  also  that  phosphorescence  can- 
not be  relied  upon  for  distinguishing  similar  water  bacteria  from 
genuine  cholera  vibrios.  Rumpel  has  reported  the  fact  that  two  un- 
doubted cultures  of  the  cholera  spirillum,  from  different  sources,  after 
being  passed  through  pigeons  and  cultivated  for  some  time  in  arti- 
ficial media,  showed  phosphorescence.  One  of  these  cultures  was  ob- 
tained originally  from  the  discharges  of  Dr.  Oergel,  who  was  a  vic- 
tim to  cholera  from  laboratory  infection  (case  reported  by  Reincke,  in 
the  Deutsche  medicinisclie  Wochenschrift,  No.  41,  1894).  Another 
case  of  supposed  laboratory  infection,  in  which  recovery  occurred,  is 
reported  by  Lazarus,  in  the  Berliner  medicinisclie  Wochenschrift9 
1893,  page  1,241. 

That  cholera  vibrios  may  be  present  in  the  alimentary  canal  of 
39 


610  PATHOGENIC   SPIRILLA. 

healthy  individuals  without  giving  rise  to  any  symptoms  of  ill-health 
appears  to  be  demonstrated.  In  support  of  this  conclusion  we  quote 
as  follows  from  a  recent  paper  by  Abel  and  Claussen: 

"  In  Wehlau  (East  Prussia),  in  the  autumn  of  1894,  seven  cases  of 
cholera  occurred  about  the  same  time.  The  members  of  the  family 
were  at  once  isolated  and  their  fa3ces  examined  almost  daily.  Of 
especial  interest  were  seventeen  individuals  who  belonged  to  families 
in  which  three  fatal  cases  occurred.  Of  these  seventeen  persons,  who 
were  not  sick  at  all  or  only  had  for  a  brief  time  a  diarrhoea,  thirteen 
had  cholera  vibrios  in  their  discharges  for  a  considerable  time.  As 
the  table  shows,  many  of  these  comma  bacilli  were  not  found  in  dis- 
charges every  day,  but  were  obtained  again  after  being  absent"  (in 
the  cultures)  "  for  a  day  or  two." 

Abel  and  Claussen  (1895),  as  a  result  of  very  extended  experi- 
ments, arrive  at  the  conclusion  that  cholera  vibrios  in  faeces  as  a  rule 
do  not  survive  longer  than  twenty  days,  and  often  cannot  be  ob- 
tained after  two  or  three  days ;  exceptionally  they  were  obtained  in 
cultures  at  the  end  of  thirty  days — Karlinsky  and  Dunbar  have  re- 
ported finding  them  at  the  end  of  fifty-two  days  and  four  months. 
Karlinsky  (1895)  has  also  reported  that  upon  woollen  and  linen  goods, 
cotton  batting  and  wool,  which  were  soaked  in  the  discharges  of 
cholera  patients  and  preserved  from  drying  by  being  wrapped  in 
waxed  paper,  the  cholera  vibrio  retained  its  vitality  for  from  twelve 
to  two  hundred  and  seventeen  days. 

The  researches  of  Kasansky  (1895)  show  that  the  cholera  spiril- 
lum is  not  destroyed  by  alow  temperature  (—30  C.)  and  that  it 
even  resists  repeated  freezing  and  thawing — three  or  four  times. 

Behring  and  Ransom  (1895)  as  a  result  of  an  extended  experi- 
mental research,  arrive  at  the  conclusion  that  cholera  cultures  from 
which  the  bacteria  have  been  removed  have  specific  toxic  properties, 
and  cause  symptoms  similar  to  those  which  result  from  the  intro- 
duction into  guinea-pigs  of  the  living  bacteria ;  that  from  these  fil- 
tered cultures  a  solid  substance  can  be  obtained  having  the  same 
toxic  properties,  and  that  from  susceptible  animals  which  have  been 
treated  with  this  toxic  substance  a  serum  can  be  obtained  which  is 
active  not  only  against  the  cholera  poison,  but  against  the  cholera 
vibrio.  These  results  support  those  previously  reached  by  other 
bacteriologists  and  lead  to  the  hope  that  a  specific  treatment  of  the 
disease  may  be  successfully  employed.  The  results  obtained  by 
Haffkine  in  India  are  favorable  to  the  view  that  his  method  of  prophy- 
laxis, by  the  subcutaneous  injection  of  virulent  cholera  cultures,  has 
a  real  value. 


PLATE   IX. 

FIG.  1. — Bacillus  diphtherias  (Klebs-Loffler)  from  culture  on  blood 
serum.  Stained  with  Loftier' s  solution  of  mechylene  blue.  x  1,000. 
Photomicrograph  by  oil  lamp.  (Borden.) 

FIG.  2. — Micrococcus  gonorrhoeas  in  urethral  pus.  Stained  with 
Loffler's  solution  of  methylene  blue,  x  1,000.  Photomicrograph  by  oil 
lamp.  (Borden.) 

FIG.  3. — Bacillus  tuberculosis  in  sputum.  x  1,000.  Photomicro- 
graph by  oil  lamp.  (Borden.) 

FIG.  4. — Bacillus  typhi  abdominalis,  from  agar  culture,  x  1,000. 
Photomicrograph  by  oil  lamp.  (Borden.) 

FIG.  5. — Streptococcus  pyogenes  (longus).  x  1,000.  Photomicro- 
graph made  at  the  Army  Medical  Museum  by  sunlight.  (Gray.) 

FIG.  6. — Bacillus  mallei,  x  1,000.  Photomicrograph  made  at  the 
Army  Medical  Museum  by  sunlight.  (Gray.) 


PLATEIX. 

STERNBERG'S  BACTERIOLOGY. 


Fig.  2. 


v». 

*  * 


i  r 


••«£#,-   H       *'l 
'    -'  rf   IT'',   »^"*    .'. 

^^^:,  JH# 


Pig.  3. 


I 

"«••!• 


.  5. 


Pigr.  4. 


<^ 

_¥     _  A     «<^ 


.  e. 


PATHOGENIC  BACTERIA. 


PART   FOURTH. 


SAPROPHYTES. 

I.  BACTERIA  IN  THE  AIR.    II.  BACTERIA  IN  WATER.    III.    BACTERIA  IN 
THE  SOIL.    IV.  BACTERIA  ON  THE  SURFACE  OF  THE  BODY  AND  OF  EX- 
POSED Mucous  MEMBRANES.    V.  BACTERIA  OF  THE  STOMACH  AND 
INTESTINE.    VI.  BACTERIA  OF  CADAVERS  AND  OF  PUTREFYING 
MATERIAL  FROM  VARIOUS  SOURCES.     VII.  BACTEBIA 
itf  ARTICLES  or  FOOD. 


I. 

BACTERIA   IX   THE   AIR. 

THE  saprophytic  bacteria  are  found  wherever  the  organic  material 
which  serves  as  their  pabulum  is  exposed  to  the  air  under  conditions 
favorable  to  their  growth.  The  essential  conditions  are  presence  of 
moisture  and  a  suitable  temperature.  The  organic  material  may  be 
in  solution  in  water  or  in  the  form  of  moist  masses  of  animal  or 
vegetable  origin,  and  the  temperature  may  vary  within  considerable 
limits — 0°  to  70°  C.  But  the  species  which  takes  the  precedence  will 
depend  largely  upon  special  conditions.  Thus  certain  species  multi- 
ply abundantly  in  water  which  contains  comparatively  little  organic 
pabulum,  and  others  require  a  culture  medium  rich  in  albuminous 
material  or  in  carbohydrates  ;  some  grow  at  a  comparatively  low  or 
high  temperature,  while  others  thrive  only  at  a  temperature  of  20°  to 
40°  C.  or  have  a  still  more  limited  range ;  some  require  an  abun- 
dant supply  of  oxygen,  and  others  will  not  grow  in  the  presence  of 
this  gas.  Our  statement  that  saprophytic  bacteria  are  found  wherever 
the  organic  material  which  serves  as  their  pabulum  is  exposed  to  the 
air — under  suitable  conditions — relates  to  the  fact  that  it  is  through 
the  air  that  these  bacteria  are  distributed  and  brought  in  contact 
with  exposed  material.  It  is  a  matter  of  common  laboratory  experi- 
ence that  sterilized  organic  liquids  quickly  undergo  putrefactive  de- 
composition when  freely  exposed  to  the  air,  and  may  be  preserved  in- 
definitely when  protected  from  the  germs  suspended  in  the  air  by 
means  of  a  cotton  air  filter.  But  the  organic  pabulum  required  for 
the  nourishment  of  these  bacteria  is  not  found  in  the  air  in  any  con- 
siderable amount,  and  if  they  ever  multiply  in  the  atmosphere  it 
must  be  under  very  exceptional  conditions.  Their  presence  is  due  to 
the  fact  that  they  are  wafted  from  surfaces  where  they  exist  in  a 
desiccated  condition,  and,  owing  to  their  levity,  are  carried  by  the 
wind  to  distant  localities.  But,  under  the  law  of  gravitation,  when 
not  exposed  to  the  action  of  currents  of  air  they  constantly  fall 
again  upon  exposed  surfaces,  which,  if  moist,  retain  them,  or  from 
which,  if  dry,  they  are  again  wafted  by  the  next  current  of  air. 
Under  these  circumstances  it  is  easy  to  understand  why,  as  deter- 


614 


BACTERIA   IX   THE   AIR. 


mined  by  investigation  more  bacteria  are  found  near  the  surface  of 
the  earth  than  at  some  distance  above  the  surface,  more  over  the 
land  than  over  the  ocean,  more  in  cities  with  their  dust-covered 
streets  than  in  the  country  with  its  grass-covered  fields. 

Careful  experiments  have  shown  that  bacteria  do  rot  find  their 
way  into  the  atmosphere  from  the  surface  of  liquids,  unless  portions 
of  the  liquid  containing  them  are  projected  into  the  air  by  some 
mechanical  means,  such  as  the  bursting  of  bubbles  of  gas.  Cultures 
of  pathogenic  bacteria  freely  exposed  to  the  air  in  laboratories  do  not 
endanger  the  health  of  those  who  work  over  them ;  but  if  such  a  cul- 
ture is  spilled  upon  the  floor  and  allowed  to  remain  without  disin- 
fection, when  it  is  desiccated  the  bacteria 
contained  in  it  will  form  part  of  the  dust  of 
the  room  and  might  be  dangerous  to  its 
occupants.  Bacteria  do  not  escape  into  the 
air  from  the  surface  of  the  fluid  contents  of 
sewers  and  cesspools,  but  changes  of  level 
may  cause  a  deposit  upon  surfaces,  which 
is  rich  in  bacteria,  and  when  dried  this  ma- 
terial is  easily  carried  into  the  atmosphere 
by  currents  of  air. 

Tyndall's  experiments  (1869)  show  that 
in  a  closed  receptacle  in  which  the  air  is 
perfectly  still  all  suspended  particles  are  af- 
ter a  time  deposited  on  the  floor  of  the  closed 
air  chamber.  And  common  experience  de- 
monstrates the  fact  that  the  dust  of  the  at- 
mosphere is  carried  by  the  wind  from  ex- 
posed surfaces  and  again  deposited  when  the 
air  is  at  rest.  This  dust  as  deposited,  for 
example,  in  our  dwellings  contains  innu- 
merable bacteria  in  a  desiccated  condition, 
and  the  smallest  quantity  of  it  introduced 
into  a  sterile  organic  liquid  will  cause  it  to 
undergo  putrefactive  decomposition,  and 
by  bacteriological  methods  it  will  be  found 
to  contain  various  species  of  bacteria.  Such 
dust  also  contains  the  spores  of  various 
mould  fungi  which  are  present  in  the  atmo- 
sphere, usually  in  greater  numbers  than  the 
bacteria.  The  mould  fungi  are  air  plants 

which  vegetate  upon  the  surface  of  moist  organic  material  and  form 
innumerable  spores,  which  are  easily  wafted  into  the  air,  both  on 
account  of  their  low  specific  gravity  and  minute  size,  and  because  they 


FIG.  186.  —  Penicillium  glau- 
cum;  m,  mycelium,  from  which 
is  given  off  a  branching  pedicle 
bearing  spores.  X  150. 


BACTERIA   IX    THE   AIR. 


Gio 


are  borne  upon  projecting  pedicles  by  which  they  are  removed  from 
the  moist  material  upon  which  and  in  which  the  mycelium  develops 
(Fig.  186),  and,  being  dry,  are  easily  carried  away  by  currents  of  air. 

Bacteriologists  have  given  much  attention  to  the  study  of  the  mi- 
croorganisms suspended  in  the  atmosphere,  with  especial  reference  to 
hygienic  questions.  The  methods  and  results  of  these  investigations 
will  be  considered  in  the  present  section. 

Pasteur  (1860)  demonstrated  the  presence  of  living  bacteria  in  the 
atmosphere  by  aspirating  a  considerable  quantity  of  air  through  a 
filter  of  gun-cotton  or  of  asbestos  contained  in  a  glass  tube.  By  dis- 
solving the  gun-cotton  in  alcohol  and  ether  he  was  able  to  demon- 
strate the  presence  of  various  microorganisms  by  a  microscopical  ex- 
amination of  the  sediment,  and  by  placing  the  asbestos  filters  in 
sterilized  culture  media  he  proved  that  living  ge.rms  had  been  filtered 
out  of  the  air  passed  through  them. 


FIG.  187. 

A  method  employed  by  several  of  the  earlier  investigators  con- 
sisted in  the  collection  of  atmospheric  moisture  precipitated  as  dew 
upon  a  surface  cooled  by  a  freezing-  mixture.  This  was  found  to  con- 
tain living  bacteria  of  various  forms.  The  examination  of  rain  water, 
which  in  falling  washes  the  suspended  particles  from  the  atmosphere, 
gave  similar  results. 

The  first  systematic  attempts  to  study  the  microorganisms  of  the 
air  were  made  by  Maddox  (1870)  and  by  Cunningham  (1873),  who 
used  an  aeroscope  which  was  a  modification  of  one  previously  de- 
scribed by  Pouchet.  In  the  earlier  researches  of  Miquel  a  similar 
aeroscope  was  used.  This  is  shown  in  Fig.  187.  The  opening  to  the 
cylindrical  tube  A  is  kept  facing  the  wind  by  means  of  a  wind  vane, 
and  when  the  wind  is  blowing  a  current  passes  through  a  small  aper- 
ture in  a  funnel-shaped  partition  which  is  properly  placed  in  the 
cylindrical  tube.  A  glass  slide,  upon  the  lower  surface  of  which  a 


616  BACTERIA   IN   THE  AIR. 

mixture  of  glycerin  and  glucose  has  been  placed,  is  adjusted  near  the 
opening  of  the  funnel,  at  a  distance  of  about  three  millimetres,  so 
that  the  air  escaping  through  the  small  orifice  is  projected  against  it. 
By  this  arrangement  a  considerable  number  of  the  microorganisms 
present  in  the  air,  as  well  as  suspended  particles  of  all  kinds,  are  ar- 
rested upon  the  surface  of  the  slide  and  can  be  examined  under  the 
microscope  or  studied  by  bacteriological  methods.  But  an  aeroscope 
of  this  kind  gives  no  precise  information  as  to  the  number  of  living 
germs  contained  in  a  definite  quantity  of  air.  The  microscopical  ex- 
amination also  fails  to  differentiate  the  bacteria  from  particles  of 
various  kinds  which  resemble  them  in  shape,  and  the  microorgan- 
isms seen  are  for  the  most  part  spores  of  various  fungi  mingled  with 
pollen  grains,  vegetable  fibres,  plant  hairs,  starch  granules,  and 
amorphous  granular  material. 

Another  method,  which  has  been  employed  by  Cohn,  Pasteur, 
Miquel,  and  others,  consists  in  the  aspiration  of  a  definite  quantity  of 
air  through  a  culture  liquid,  which  is  then  placed  in  an  incubating 
oven  for  the  development  of  microorganisms  washed  out  of  the  air 
which  has  been  passed  through  it.  This  method  shows  that  bacteria 
of  different  species  are  present,  but  gives  no  information  as  to  their 
relative  number,  and  requires  further  researches  by  the  plate  method 
to  determine  the  characters  of  the  several  species  in  pure  cultures. 

A  far  simpler  method  consists  in  the  exposure  of  a  solid  culture 
medium,  which  has  been  carefully  sterilized  and  allowed  to  cool  on  a 
glass  plate  or  in  a  Petri's  dish,  for  a  short  time  in  the  air  to  be  ex- 
amined. Bacteria  and  mould  fungi  deposited  from  the  air  adhere  to 
the  surface  of  the  moist  culture  medium,  and  form  colonies  when  the 
plate,  enclosed  in  a  covered  glass  dish,  is  placed  in  the  incubating  oven. 
The  number  of  these  colonies  which  develop  after  exposure  in  the 
air  for  a  given  time  enables  us  to  estimate  in  a  rough  way  the  num- 
ber of  microorganisms  present  in  the  air  of  the  locality  where  the 
exposure  was  made  ;  and  the  variety  of  species  is  determined  by  ex- 
amining the  separate  colonies,  each  of  which  is,  as  a  rule,  developed 
from  a  single  germ.  By  exposing  a  number  of  plates  at  different 
times  this  method  enables  us  to  determine  what  species  are  most 
abundant  in  a  given  locality  and  the  comparative  number  in  dif- 
ferent localities,  as  determined  by  counting  the  colonies  after  ex- 
posure for  a  definite  time — e.g.,  ten  minutes.  Of  course  we  will  only 
obtain  evidence  of  the  presence  of  such  aerobic  bacteria  as  will 
grow  in  our  culture  medium.  The  anaerobic  bacteria  may  be  studied 
by  placing  plates  exposed  in  a  similar  way  in  an  atmosphere  of  hydro- 
gen. Bacteria  which  grow  slowly  and  only  under  special  conditions, 
like  the  tubercle  bacillus,  would  be  likely  to  escape  observation,  as 
the  mould  fungi  and  common  saprophytes  would  take  complete  pos- 


BACTERIA  IN   THE   AIR. 


617 


session  of  the  surface  of  the  culture  medium  before  the  others  had 
formed  visible  colonies.  Students  will  do  well  to  employ  this  simple 
and  satisfactory  method  for  the  purpose  of  making  themselves  familiar 
with  the  more  common  atmospheric  organisms,  and  they  will  find 
the  shallow  glass  dishes  with  a  cover,  known  as  Petri's  dishes,  very 
convenient  for  the  purpose.  These  dishes  should  be  sterilized  in  the 
hot-air  oven  and  sufficient  sterile  nutrient  gelatin  or  agar  poured 
into  them  to  cover  the  bottom.  After  the  culture  medium  has  be- 
come solid  by  cooling,  the  exposure  may  be  made  by  simply  remov- 
ing the  cover  and  replacing  it  at  the  end  of  the  time  fixed  upon. 


FIG.  188. 

To  determine  in  a  more  exact  way  the  number  of  microorganisms 
contained  in  a  given  quantity  of  air  will  require  other  methods.  But 
we  may  say,  en  passant,  that  such  a  determination  is  usually  not  of 
great  scientific  importance.  The  number  is  subject  to  constant  fluc- 
tuations in  the  same  locality,  depending  upon  the  force  and  direction 
of  the  wind.  If  we  have  on  one  side  of  our  laboratory  a  dusty 
street  and  on  the  other  a  green  field,  more  bacteria  will  naturally  be 
found  when  the  wind  blows  from  the  direction  of  the  street  than 
when  it  comes  from  the  opposite  direction  ;  or,  if  the  air  is  filled  with 
dust  from  recently  sweeping  the  room,  we  may  expect  to  find  very 


CIS  BAfTKKIA   IX    THK   AIR. 

many  more  than,  when  the  room  has  been  undisturbed  for  some  time. 
The  painstaking  researches  which  have  already  been  made  have  es- 
tablished in  a  general  way  the  most  important  facts  relating  to  the 
distribution  of  atmospheric  bacteria,  but  have  failed  to  show  any  de- 
finite relation  between  the  number  of  atmospheric  bacteria  and  the 
prevalence  of  epidemic  di  .In  the  apparatus  of  Hesse,  Fig. 

188,  a  glass  tube,  having  a  diameter  of  four  to  five  centimetres  and  a 
length  of  half  a  metre  to  a  metre,  is  employed.  In  use  this  is  sup- 
ported upon  a  tripod,  as  shown  in  the  figure,  and  air  is  drawn 
through  it  by  a  wat'-r  aspirator  consisting  of  two  flasks,  also  shown. 
The  upper  flask  being  filled  with  water,  this  flows  into  the  lower 
flask  by  siphon  action,  and  upon  reversing  the  position  of  the  flasks 
number  one  is  again  filled.  By  repeating  this  operation  as  many 
times  as  desired  a  quantity  of  air  corresponding  with  the  amount  of 
water  passed  from  the  upper  to  the  lower  flask  is  slowly  aspirated 
through  the  horizontal  glass  tube.  The  microorganisms  present  are 
d< -pi^ited  upon  nutrient  gelatin  previously  allowed  to  cool  upon  the 
lower  portion  of  the  large  glass  tube.  The  air  enters  through  a  small 
opening  in  a  piece  of  sheet  rubber  which  is  tied  over  the  extremity 
of  the  horizontal  tube,  and  before  the  aspiration  is  commenced  this 
opening  is  covered  by  another  piece  of  sheet  rubber  tied  over  the 
first.  Experience  shows  that  when  the  air  is  slowly  aspirated  most 
of  the  germs  contained  in  it  are  <  i  t  ed  near  the  end  of  the  tube 
through  \vhich  it  enters.  The  colonies  which  develop  upon  the  nu- 
trient gelatin  show  the  number  and  chara<-t;-r  of  living  microorgan- 
<  -o  1 1 1 ;  i  i  1 1  ed  in  the  measured  quantity  of  air  aspirated  through  the 

appacaf  us.      Tin-  method  with  a  soluble  filter  of   ptil  v.-ri/rd    sugar,  to 

be  described  hereafter,  is  preferable  when  exact  results  are  desired; 
and  for  the  purpose  of  determining  the  relative  abundance  and  the 
variety  of  microorganisms  present  in  the  atmosphere  of  a  given  lo- 
cality the  exposure  of  nutrient  gelatin  in  Potri's  dishes  is  far  simpler, 
and,  as  a  nil.-,  will  furnish  all  the  information  that  is  of  real 
value. 

In  his  extended  researches  made  at  the  laboratory  of  Montsouri, 
in  Paris,  Miquel  has  used  various  forms  of  apparatus  and  has  ob- 
tained interesting  results  ;  but  his  method  of  ensemencements  frac- 

//o// //As  requires  a  great  expenditure  of  time  and  patience,  and  the 
more  recent  method  with  soluble  filters  is  to  be  preferred. 

In  his  latest  modification  of  the  m-;  hod  rrfn-rod  to  Miquel  used  a 
flask  like  that  shown  in  Fig.  189.  From  twenty  to  forty  cubic  cen- 
1  i  metres  of  distilled  water  are  introduced  into  this  flask.  The  cap  A 
contains  a  cotton  air  filter  and  is  fitted  to  the  neck  of  the  flask  by  a 

-•n.iind  John.  This  is  removed  during  t'x>  r\ prrimml.  The  tube  (' 
is  ronnertrd  with  a-  irator.  It  Contains  two  Cotton  Or  asbestos 


K.U TKR1A    IX    THK    A  IK. 


619 


FIG. 


filters,  c  and  b.  The  cap  being-  removed  and  the  aspirator  attached, 
the  air  is  drawn  through  the  water,  by  which  suspended  germs  are 
arrested  :  or  if  not  they  are  caught  by  the  inner  cotton  ping  b.  The 
led  point  of  the  tube  B  is  now  broken  oft*,  and  the  contents  of  the 
tlask  equally  divided  in  thirty  to  forty  tubes  containing  bouillon, 
which  are  placed  in  the  incubating  oven. 
Twenty-five  cubic  centimetres  of  bouillon 
are  also  introduced  into  the  tlask.  and  the 
cotton  plug  />  is  pushed  into  it  so  that  any 
bacteria  arrested  by  it  may  develop.  If 
one-fourth  or  one-tifth  of  the  bouillon  tubes 
show  a  development  of  bacteria  it  is  in- 
ferred that  each  culture  originated  from 
a  single  germ,  a*ul  the  number  present  in 
the  amount  of  air  drawn  through  the  tlask 
i-  estimated  from  the  number  of  tubes  in 
which  development  occurs. 

The  method  adopted  by  Straus  and  \Viirt /.  is  more  convenient  and 
more  reliable  in  its  results.  This  consists  in  passing  the  air  by  means 
of  an  aspirator  through  liquefied  nutrient  gelatin  or  agar.  The  ap- 
paratus shown  in  Fig.  1W  is  used  for  this  purpose.  Two  cotton 
plugs  are  placed  in  the  tube  B,  to  which  the  aspirator  is  attached, 
and  after  the  determined  |iiantity  of  air  has  been  passed  through  the 
liquefied  medium  the  inner  plug  is  pushed  down  with  a  sterili/.ed 

platinum  needle  so  as  to  wash  out  in  the  culture 
medium  any  germs  arrested  by  it.  Finally  the 
gelatin  or  agar  is  solidified  upon  the  walls  of 
the  tube  A  by  rotating  it  upon  a  block  of  ice  or 
under  a  stream  of  cold  water.  It  is  now  put 
aside  for  the  development  of  colonies,  which  are 
counted  to  determine  the  number  of  germs  pre- 
sent in  the  quantity  of  air  passed  through  the 
liquefied  culture  medium.  The  main  difficulty 
with  this  apparatus  is  found  in  the  fact  that  the 
nutrient  gelatin  foams  when  air  is  bubbled 
through  it  ;  for  this  reason  an  agar  medium  is 
to  be  preferred.  In  using  this  it  will  be  neces- 
sary to  place  the  liquefied  agar  in  a.  bath  main- 
tained at  W  C.  Foaming  of  the  gelatin  is  pre- 
vented by  adding  a  drop  of  olive  oil  before  ster- 
ili/.ation  in  the  steam  sterili/.or.  But  this  inter- 
feres with  the  transparency  of  the  medium. 
In  the  earlier  experiments  upon  atmospheric  organisms  Pasteur 
used  a  filter  of  asbestos,  which  was  subsequently  washed  out  in  a 


FlO.   ISHI. 


620 


BACTERIA   IN   THE   AIR. 


culture  liquid.  A  filter  of  this  kind  washed  out  in  liquefied  gelatin 
or  nutrient  agar  would  give  more  satisfactory  results,  as  the  culture 
medium  could  be  poured  upon  plates  or  spread  upon  the  walls  of  a 
test  tube  and  the  colonies  counted  in  the  usual  way.  Petri  prefers 
to  use  a  filter  of  sand,  which  he  finds  by  experiment  arrests  the  mi- 
croorganisms suspended  in  the  atmosphere,  and  which  is  subsequently 
distributed  through  the  culture  medium.  The  sand  used  is  such  as 

has  been  passed  through  a  wire  sieve  having 
openings  of  0. 5  millimetre  in  diameter.     This  is 
sterilized  by  heat,  and  is  supported  in  a  cylin- 
drical glass  tube  by  small  wire-net  baskets.    The 
complete  arrangement  is  shown  in  Fig.    191. 
Two  sand  filters,  c,  and  C2,  are  used,  the  lower 
one  of  which  serves  as  a  control  to  prove  that 
all  microorganisms  present  in  the  air  have  been 
arrested  by  the  upper  one.     The  upper  filter  is 
protected,  until  the  aspirator  attached  to  the 
tube  h  is  put  in  operation,  by  a  sterile  cotton 
plug,  not  shown  in  the  figure  which  represents 
the  filter  in  use.    Petri  uses  a  hand  air  pump  as 
an  aspirator,  and  passes  one  hundred  litres  of 
air  through  the  sand  in  from  ten  to  twenty 
minutes.     The  sand  from  the  two  filters  is  then 
distributed  in  shallow  glass  dishes  and  liquefied 
gelatin  is  poured  over  it ;  this  is  allowed  to  sol- 
idify and  is  put  aside  for  the  development  of 
colonies.    The  principal  objection  to  this  method 
is  the  presence  of  the  opaque  particles  of  sand 
in  the  culture  medium.    This  objection  has  been 
overcome  by  the  use  of  soluble  filters,  a  method 
first  employed  by  Pasteur  and  since  perfected 
by  Sedgwick  and  by  Miquel.     The  most  useful 
material  for  the  purpose  appears  to  be  cane 
sugar,  which  can  be  sterilized  in  the  hot-air  oven 
at  150°  C.  without  undergoing  any  change  in 
its  physical  characters.     Loaf  sugar  is  pulver- 
ized in  a  mortar  and  passed  through  two  sieves 
in  order  to  remove  the  coarser  grains  and  the 
very  fine  powder,  leaving  for  use  a  powder  having  grains  of  about 
one-half  millimetre  in   diameter.     This  powdered  sugar   is  placed 
in  a  glass  tube  provided  with  a  cap  having  a  ground  joint  and  a  cot- 
ton plug  to  serve  as  an  air  filter  (A,  Fig.  192),  or  in  a  tube  such  as  is 
shown  at  B,  having  the  end  drawn  out  and  hermetically  sealed.     Two 
cotton  plugs  are  placed  at  the  lower  portion  of  the  tube,  at  a  and  at  b. 


FIG.  191. 


BACTERIA   IN   THE   AIR. 


621 


Glass  tubing  having  a  diameter  of  about  five  millimetres  is  used  in 
making  these  tubes,  and  from  one  to  two  grammes  of  powdered  sugar 
is  a  suitable  quantity  to  use  as  a  filter.  The  whole  apparatus  is  steril- 
ized for  an  hour  at  150°  C.  in  a  hot-air  oven  after  the  pulverized 
sugar  has  been  introduced.  Before  using  it  will  be  necessary  to 
pack  the  sugar  against  the  supporting  plug  a  by  gently  striking  the 
lower  end  of  the  tube,  held  in  a  vertical  position,  upon  some  horizon- 
tal surface ;  and  during  aspiration 
the  tube  must  remain  in  a  vertical 
position,  or  nearly  so,  in  order  that 
the  sugar  may  properly  fill  its  entire 
calibre.  The  aspirator  is  attached  to 
the  lower  end  of  the  tube  by  a  piece 
of  rubber  tubing.  When  the  tube  B 
is  used  the  sealed  extremity  is  broken 
off  at  the  moment  that  the  aspirator 
is  set  in  action,  and  it  is  again  sealed 
in  a  flame  after  the  desired  amount 
of  air  has  been  passed  through  the 
filter.  The  next  step  consists  in  dis- 
solving the  sugar  in  distilled  water 
or  in  liquefied  gelatin.  To  insure 
the  removal  of  all  the  sugar  the  cot- 
ton plug  a  may  be  pushed  out  with  a 
sterilized  glass  rod,  after  removing  b 
with  forceps.  From  fifty  to  five  hun- 
dred cubic  centimetres  of  distilled 
water,  contained  in  an  Erlenmeyer 
flask  and  carefully  sterilized,  may  be 
used,  the  amount  required  depending 
upon  circumstances  relating  to  the 
conditions  of  the  experiment.  By 
adding  five  or  ten  cubic  centimetres 
of  this  water,  containing  the  sugar 
and  microorganisms  arrested  by  it, 
to  nutrient  gelatin  or  agar  liquefied  by  heat,  and  then  making  Es- 
march  roll  tubes,  the  number  of  germs  in  the  entire  quantity  is  easily 
estimated  by  counting  the  colonies  which  develop  in  the  roll  tubes. 

Sedgwick  and  Tucker,  in  a  communication  made  to  the  Boston 
Society  of  Arts,  January  12th,  1888,  were  the  first  to  propose  the  use 
of  a  soluble  filter  of  granulated  sugar  for  collecting  atmospheric 
germs.  Their  complete  apparatus  consists  of  an  exhausted  receiver, 
from  which  a  given  quantity  of  air  is  withdrawn  by  means  of  an  air 
pump.  A  vacuum  gauge  is  attached  to  the  receiver,  which  is  coupled 


FIG.  192. 


FIG.  193. 


G22  BACTERIA    IX   THE   AIR. 

with  the  glass  tube  containing  the  granulated -sugar  filter  by  a  piece 
of  rubber  tubing.  Instead  of  transferring  the  soluble  filter  to  gela- 
tin in  test  tubes,  they  use  a  large  glass  cylinder  having  a  slender 
stem,  in  which  the  sugar  is  placed  (Fig.  193).  After  the  aspiration 
liquefied  gelatin  is  introduced  into  the  large  glass  cylinder,  which  is 
held  in  a  horizontal  position  ;  the  sterilized  cotton  plug  is  then  re- 
placed in  the  mouth  of  the  cylinder,  the  sugar  is  pushed  into  the 
liquefied  gelatin  and  dissolved,  and  by  rotating  the  cylinder  upon  a 
block  of  ice  the  gelatin  is  spread  upon  its  walls  as  in  an  Esmarch  roll 
tube.  For  convenience  in  counting  the  colonies  lines  are  drawn  upon 
the  surface  of  the  cylinder,  dividing  it  into  squares  of  uniform  di- 
mensions. 


GENERAL  RESULTS   OF  RESEARCHES   MADE. 

As  already  stated,  the  presence  of  bacteria  in  the  atmosphere  de- 
pends upon  their  being  wafted  by  currents  of  air  from  surfaces  where 
they  are  present  in  a  desiccated  condition.  That  they  are  not  carried 
away  from  moist  surfaces  is  shown  by  the  fact  that  expired  air  from 
the  human  lungs  does  not  contain  microorganisms,  although  the  in- 
spired air  may  have  contained  considerable  numbers,  and  there  are 
always  a  vast  number  present  in  the  salivary  secretions.  The  moist 
mucous  membrane  of  the  respiratory  passages  constitutes  a  germ 
trap  which  is  much  more  efficient  than  the  glass  slide  smeared  with 
glycerin  used  in  some  of  the  aeroscopes  heretofore  described,  for  it 
is  a  far  more  extended  surface.  As  a  matter  of  fact,  most  of  the  sus- 
pended particles  in  inspired  air  are  deposited  before  the  current  of 
air  passes  through  the  larynx. 

Air  which  passes  over  large  bodies  of  water  is  also  purified  of  its 
germs  and  other  suspended  particles.  The  researches  of  Fischer 
show  that  at  a  considerable  distance  from  the  land  no  germs  are 
found  in  the  atmosphere  over  the  ocean,  and  that  it  is  only  upon  ap- 
proaching land  that  their  presence  is  manifested  by  the  development 
of  colonies  upon  properly  exposed  gelatin  plates. 

Uffelmann  found,  in  his  researches,  that  in  the  open  fields  the 
number  of  living  germs  in  a  cubic  metre  of  air  averaged  two  hundred 
and  fifty,  on  the  sea  coast  the  average  was  one  hundred,  in  the  court- 
yard of  the  University  of  Rostock  four  hundred  and  fifty.  The  num- 
ber was  materiallv  reduced  after  a  rainfall  and  increased  when  a 
dry  land  wind  prevailed. 

Frankland  fonnd  that  fewer  germs  were  present  in  the  air  in 
winter  than  in  summer,  and  that  when  the  earth  was  covered  with 
snow  the  number  was  greatly  reduced,  as  also  during  a  light  fall  of 
snow  ;  the  air  of  towns  was  found  to  be  more  rich  in  germs  than  the 


BACTERIA    IX   THE   AIR. 

air  of  the  country ;  the  lower  strata  of  the  atmosphere  contained 
more  than  the  air  of  elevated  localities. 

Von  Freudenreich  also  found  that  the  air  of  the  country  contained 
fewer  germs  than  that  of  the  city.  Thus  in  the  city  of  Berne  a  cubic 
metre  of  air  often  contained  as  many  as  two  thousand  four  hundred 
germs,  while  the  maximum  in  country  air  was  three  hundred.  His  re- 
sults corresponded  with  those  of  Miquel  in  showing  that  the  number 
of  atmospheric  organisms  is  greater  in  the  morning  and  the  evening, 
between  the  hours  of  6  and  8,  than  during  the  rest  of  the  day.  Neu- 
mann, whose  researches  were  made  in  the  Moabite  Hospital,  found 
the  greatest  number  of  bacteria  in  the  air  in  the  morning  after  the 
patients  able  to  sit  up  had  left  their  beds  and  the  wards  had  been 
swept.  The  number  of  germs  was  then  from  eighty  to  one  hundred 
and  forty  in  ten  litres  of  air,  while  in  the  evening  the  number  fell  to 
four  to  ten  germs  in  ten  litres. 

Miquel  has  given  the  following  summary  of  results  obtained  in 
his  extended  experiments,  made  in  Paris  during  the  years  1881,  1882, 
and  1883  : 


Average  for 
«        « 

«        « 

1880  

Number  of  Germs  in  a  Cubic  Metre  of  Air. 

Air  of  Laboratory, 
Montsouri. 

Air  of  Park,  Mont 
souri. 

215 
348 
550 

71 
62 
51 

1881  

1882.. 

Rue  de  Rivoli,  average  for  one  year,  750  ;  summit  of  Pantheon,  28  ; 
Hotel-Dieu,  1880,  average  for  four  months,  male  ward  6,300,  female 
ward  5,120  ;  La  Piete  Hospital,  average  of  fifteen  months,  11,100. 

It  must  be  remembered  that  the  figures  given  relate  both  to  bac- 
teria and  to  the  spores  of  mould  fungi,  and  that  the  latter  are  com- 
monly the  most  numerous  when  the  experiment  is  made  in  the  open 
air.  Petri  has  shown  that  when  gelatin  plates  are  exposed  in  the  air 
the  relative  number  of  spores  of  mould  fungi  deposited  upon  them  is 
less  than  is  obtained  in  aspiration  experiments. 

The  number  of  colonies  which  develop  on  exposed  plates  does  not 
represent  the  full  number  of  bacteria  deposited,  for  these  colonies 
very  frequently  have  their  origin  in  a  dust  particle  to  which  several 
bacteria  are  attached,  or  in  a  little  mass  of  organic  material  contain- 
ing a  considerable  number. 

It  is  generally  conceded  that  sea  air  and  country  air  are  more 
wholesome  than  the  air  of  cities,  and  especially  of  crowded  apart- 
ments, in  which  the  number  of  bacteria  has  been  shown  to  be  very 
much  greater.  But  it  would  be  a  mistake  to  ascribe  the  sanitary 
value  of  sea,  country,  and  mountain  air  to  the  relatively  small  num- 


624  BACTERIA   IN   THE   AIR. 

her  of  bacteria  present  in  such  air.  There  are  other  important  fac- 
tors to  be  considered,  and  we  have  no  satisfactory  evidence  that  the 
number  of  saprophytic  bacteria  present  in  the  air  has  an  important 
bearing  upon  the  health  of  those  who  respire  it.  We  do  know  that 
the  confined  air  of  crowded  apartments,  and  especially  of  factories 
in  which  a  large  quantity  of  dust  is  suspended  in  the  air,  predisposes 
those  breathing  such  air  to  pulmonary  diseases  and  lowers  the  gen- 
eral standard  of  health.  But  it  has  not  been  proved  that  this  is  due 
to  the  presence  of  bacteria.  Infectious  diseases  may,  under  certain 
circumstances,  be  communicated  by  way  of  the  respiratory  passages 
as  a  result  of  breathing  air  containing  in  suspension  pathogenic  bac- 
teria ;  but  there  is  reason  to  believe  that  this  occurs  less  frequently 
than  is  generally  supposed. 

Kruger  has  shown  that  the  dust  of  a  hospital  ward  in  which  pa- 
tients with  pulmonary  consumption  expectorated  occasionally  upon 
the  floor  contained  tubercle  bacilli.  This  was  proved  by  wiping  up 
the  dust  on  a  sterilized  sponge,  washing  this  out  in  bouillon,  and  in- 
jecting this  into  the  cavity  of  the  abdomen  of  guinea-pigs.  Two 
animals  out  of  sixteen  injected  became  tuberculous.  In  pulmonic 
anthrax,  which  occasionally  occurs  in  persons  engaged  in  sorting 
wool — "wool-sorters'  disease" — infection  occurs  as  a  result  of  the 
respiration  of  air  containing  the  spores  of  the  anthrax  bacillus. 

Among  the  non-pathogenic  saprophytes  iound  in  the  air  certain 
aerobic  micrococci  appear  to  be  the  most  abundant,  and,  as  a  rule, 
bacilli  are  not  found  in  great  numbers  or  variety.  In  some  localities 
various  species  of  sarcinse  are  especially  abundant.  The  following 
is  a  partial  list  of  the  species  which  have  been  shown  by  the  researches 
of  various  bacteriologists  to  be  occasionally  present  in  the  air.  But, 
as  heretofore  remarked,  their  presence  is  to  be  regarded  as  acci- 
dental, and  so  far  as  we  know  there  is  no  bacterial  flora  properly  be- 
longing to  the  atmosphere  : 

Micrococcus  urese  (Pasteur),  Diplococcus  rosens  (Bumm),  Diplococcus 
citreus  conglomeratus  (Bumm),  Micrococcus  radiatus  (Fliigge),  Micrococcus 
flavus  desidens  (Fliigge),  Micrococcus  flavus  liquefaciens  (Fliigge),  Micro- 
coccus  tetragenus  versatilis  (Sternberg),  Micrococcus  pyogenes  aureus  (Rosen- 
bach),  Micrococcus  pyogenes  citreus  (Passet),  Micrococcus  cinnabareus 
(Fliigge),  Micrococcus  flavus  tardigradus  (Fliigge),  Micrococcus  versicolor 
(Fliigge),  Micrococcus  viticulosus  (Katz),  Micrococcus  candidans  (Fliigge), 
Pediococcus  cerevisias  (Balcke),  Sarcina  lutea  (Schroter),  Sarcina  rosea 
(Schroter),  Sarcina  aurantiaca,  Sarcina  alba,  Sarcina  Candida  (Reinke), 
Bacillus  tumescens  (Zopf),  Bacillus  subtilis  (Ehrenberg),  Bacillus  multipedi- 
culosus  (Fliigge),  Bacillus  mesentericus  fuscus  (Fliigge),  Bacillus  mesenteri- 
cus  ruber  (Globig),  Bacillus  inflatus  (A.  Koch),  Bacillus  mesentericus  vul- 
gatus,  Bacillus  prodigiosus,  Bacillus  aerophilus  (Liborius),  Bacillus  pestifer 
(Frankland),  Spirillum  aureum  (Weibel),  Spirillum  flavescens  (Weibel),  Spi- 
rillum flavum  (Weibel),  Bacillus  Havaniensis  (Sternberg). 

In  the  researches  of  Welz,  made  in  the  vicinity  of  Freiburg,  twenty- 
three  different  micrococci  and  twenty-two  bacilli  were  obtained  from  the 
air. 


BACTERIA   IX   THE   AIR.  625 


ADDITIONAL   NOTES   UPON   BACTERIA  IN   THE   AIR. 

Ruete  and  Enoch  (1895)  have  examined  the  air  of  closed  schoolrooms 
with  the  following  results.  Eighteen  different  species  were  obtained,  only 
one  of  which  proved  to  be  pathogenic  for  mice,  guinea-pigs,  and  rabbits.  The 
number  of  bacteria  per  cubic  metre  varied  from  1,500  to  3,000,000,  the  aver- 
age being  about  268,000.  The  observations  were  made  during  the  winter 
months. 

Marpmaiin  (1893),  in  his  examination  of  dust  collected  in  the  streets  of  Leip- 
zig for  tubercle  bacilli,  obtained  positive  results  from  a  considerable  pro- 
portion of  the  specimens  examined.  Evidently  these  bacilli  in  dust  from  the 
streets  are  liable  to  be  blown  into  the  air  and  deposited  upon  the  mucous 
membrane  of  the  respiratory  passages  of  those  breathing  this  air.  Christian! 
(1893)  has  shown  that,  as  a  rule,  no  bacteria  are  present  in  the  air  at  an  alti- 
tude of  one  thousand  metres  or  more  above  the  soil  (air  collected  during 
balloon  ascensions). 

Dyar  (1895)  has  made  a  careful  study  of  the  microorganisms  found  in.  the 
air  in  the  city  of  New  York.  He  has  described  numerous  species  of  micro- 
cocci  and  bacilli  found  chiefly  in  the  air  of  the  hallway  of  the  College  of 
Physicians  and  Surgeons.  Some  of  these  are  new  and  some  have  been 
identified  as  previously  described  species. 

40 


II. 

BACTERIA  IN  WATER. 

THE  water  of  the  ocean,  of  lakes,  ponds,  and  running  streams 
necessarily  contains  bacteria,  as  they  are  constantly  being  carried 
into  it  by  currents  of  air  passing  over  the  neighboring  land  surfaces, 
and  by  rain  water  which  washes  suspended  microorganisms  from 
the  atmosphere  ;  and,  as  such  water  contains  more  or  less  organic 
material  in  solution,  many  of  the  saprophytic  bacteria  multiply  in  it 
abundantly.  It  is  only  in  the  water  of  springs  and  wells  which 
comes  from  the  deeper  strata  of  the  soil  that  they  are  absent.  The 
number  and  variety  of  species  present  in  water  from  any  given 
source  will  depend  upon  conditions  relating  to  the  amount  of  organic 
pabulum,  the  temperature,  the  depth  of  .the  water,  the  fact  of  its 
being  in  motion  or  at  rest,  its  pollution  from  various  sources,  etc. 
The  comparatively  pure  water  of  lakes  and  running  streams  contains 
a  considerable  number  of  bacteria  which  find  their  normal  habitat 
in  such  waters  and  which  multiply  abundantly  in  them,  notwith- 
standing the  small  quantity  of  organic  matter  and  salts  which  they 
contain.  The  water  of  stagnant,  shallow  pools,  and  of  sluggish 
streams  into  which  sewage  is  discharged,  contains  a  far  greater 
number  and  a  greater  variety  of  species. 

The  study  of  these  bacteria  in  water  has  received  much  attention 
on  account  of  the  sanitary  questions  involved,  relating  to  the  use  of 
water  from  various  sources  for  drinking  purposes.  In  the  present 
section  we  shall  first  give  an  account  of  the  methods  of  bacteriologi- 
cal water  analysis,  and  then  a  condensed  statement  of  results  ob- 
tained in  the  very  numerous  investigations  which  have  been  made. 

A  very  important  point  to  be  kept  in  view  is  the  fact  that  a  great 
increase  in  the  number  of  bacteria  present,  in  samples  of  water  col- 
lected for  investigation,  is  likely  to  occur  if  these  samples  are  kept 
for  some  time.  A  water  which,  for  example,  contains  only  two 
hundred  to  three  hundred  bacteria  per  cubic  centimetre  when  the  ex- 
amination is  made  at  once,  may  contain  several  thousand  at  the  end 
of  twenty-four  hours,  and  at  the  end  of  the  second  or  third  day 
twenty  thousand  or  more  may  be  present  in  the  same  quantity. 


BACTERIA    IX   WATER.  627 

Later,  on  account  of  the  exhaustion  of  organic  pabulum,  the  num- 
ber is  again  reduced  as  the  bacteria  present  gradually  lose  their 
vitality.  Under  these  circumstances  it  is  evident  that  an  estimate  of 
the  number  of  bacteria  present  in  water  from  a  given  source  can 
have  no  value,  unless  a  sample  is  tested  by  bacteriological  methods 
within  a  short  time  after  it  has  been  collected.  Not  more  than  an 
hour  or  two  should  be  allowed  to  elapse,  especially  in  warm  weather. 
By  placing  the  water  upon  ice  the  time  may  be  extended  somewhat, 
but  Wolffhiigel  has  shown  that  the  number  of  germs  is  gradually 
diminished  when  water  is  preserved  in  this  way,  and  it  will  be  safest 
to  make  an  immediate  examination  when  this  is  practicable. 

The  collection  may  be  made  in  a  sterilized  Erlenmeyer  flask  pro- 
vided with  a  cotton  air  filter,  or  in  a  bottle  having  a  ground-glass 
stopper  which  has  been  wrapped  in  tissue  paper  and  sterilized  for  an 
hour  or  more  at  150°  C.  in  the  hot-air  oven.  Or  the  small  flasks  with 
a  long  neck  may  be  used,  as  first  recommended  by  Pasteur.  These 
are  prepared  as  follows  :  The  bulb  is  first  gently  heated,  and  the  ex- 
tremity of  the  tube  dipped  into  distilled  water,  which  mounts  into 


FIG.  1W. 

the  bulb  as  it  cools ;  the  water  is  then  made  to  boil,  and  when  all 
but  a  drop  or  two  has  escaped  and  the  bulb  is  filled  with  steam  the 
extremity  of  the  tube  is  hermetically  sealed.  When  the  steam  has 
condensed  by  the  cooling  of  the  bulb  a  partial  vacuum  is  formed, 
and  the  tube  is  ready  for  use  at  any  time.  It  is  filled  with  water  by 
breaking  off  the  sealed  extremity  under  the  surface  of  the  water  of 
which  a  sample  is  desired.  This  is  done  with  sterilized  forceps,  and 
care  must  be  taken  that  the  exterior  of  the  tube  is  properly  sterilized 
before  the  collection  is  made.  The  end  is  immediately  sealed  in  the 
flame  of  a  lamp.  A  difficulty  with  these  vacuum  tubes  is  that  they 
are  so  completely  filled  with  water  that  this  cannot  be  readily  drawn 
from  them  again  in  small  quantities.  The  writer  therefore  prefers 
to  make  the  collection  in  a  tube  shaped  as  shown  in  Fig.  194,  in  which 
a  partial  vacuum  is  formed  just  before  the  collection  by  heating  tho 
air  in  the  bulb.  The  water  mounts  into  the  tube  as  the  air  in  tho 
bulb  cools,  and  is  readily  forced  out  again  for  making  cultures  by 
applying  gentle  heat  to  the  bulb.  As  a  lamp  is  needed  to  seal  the  end 
of  the  tube  in  either  case,  there  is  no  special  advantage  in  having  a 
vacuum  formed  in  advance,  and,  as  stated,  the  vacuum  tubes  are  so 


628 


BACTERIA    IX    WATER. 


nearly  filled  with  water  that  it  is  not  so  simple  a  matter  to  obtain  the 
contents  for  our  culture  experiments  without  undue  exposure  to  at- 
mospheric germs.  In  practice  small  glass  bottles  with  ground-glass 
stoppers  will  be  found  most  convenient,  and,  when  properly  steril- 
ized, are  unobjectionable.  They  should  be  filled  at  a  little  distance 
below  the  surface,  as  there  is  often  a  deposit  of  dust  upon  the  surface 

of  standing  water,  and  sometimes  a 
delicate  film  made  up  of  aerobic  bac- 
teria. When  water  is  to  be  obtained 
from  a  pump  or  a  hydrant  it  should 
be  allowed  to  flow  for  some  time  before 
the  collection  is.  made.  To  collect 
water  at  various  depths  the  apparatus 
shown  in  Fig.  195  is  recommended  by 
Lepsius.  An  iron  frame  supports  an 
inverted  flask,  A,  filled  with  sterilized 
mercury  and  containing  about  three 
hundred  cubic  centimetres.  The  flask 
B  is  intended  to  receive  the  mercury 
when,  at  the  desired  depth,  it  is  al- 
lowed to  flow  through  the  capillary 
tube  b.  This  is  sealed  at  the  extremity 
and  bent  as  shown  in  the  figure.  By 
pulling  upon  the  cord  c  this  tube  is 
broken,  and  as  the  mercury  flows  from 
the  flask  this  is  filled  with  water 
through  the  tube  a.  The  extremity 
of  the  broken  tube  b  is  closed  by  the 
mercury  in  the  flask  B  when  A  is  full 
of  water,  and  the  apparatus  can  be 
brought  to  the  surface  with  only  such 
water  as  was  collected  at  the  depth 
from  which  a  sample  was  desired. 

The  bacteriological  analysis  is 
made  by  adding  a  definite  quantity 
of  the  water  under  investigation  to 
liquefied  gelatin  or  agar-gelatin,  and 
making  a  plate  or  Esmarch  roll  tube,  which  is  put  aside  for  the  devel- 
opment of  colonies.  Miquel  and  others  have  preferred  to  use  liquid 
cultures  and  the  method  of  fractional  cultivation  described  in  the 
previous  section.  The  use  of  a  solid  culture  medium  has,  however, 
such  obvious  advantages  that  we  do  not  consider  it  necessary  to  do 
more  than  refer  to  the  other  method  as  one  which,  when  applied 
with  skill  and  patience,  may  give  sufficiently  accurate  results. 


FK,.  1 1 


BACTERIA   IX    WATER.  629 

The  amount  of  water  which  should  be  added  to  the  usual  quan- 
tity of  liquefied  flesh-peptone-gelatin  in  a  test  tube,  in  order  that  the 
colonies  which  develop  may  be  well  separated  from  each  other  and 
easily  counted,  can  only  be  determined  by  experiment.  If  the  water 
is  from  an  impure  source  a  single  drop  may  be  too  much,  and  it  will 
be  necessary  to  dilute  it  with  distilled  water  recently  sterilized.  But 
for  ordinary  potable  water  it  will  usually  be  best,  in  a  first  experi- 
ment, to  make  two  trials,  one  with  one  cubic  centimetre  and  one 
with  one-half  cubic  centimetre  added  to  the  liquefied  nutrient  gelatin. 
The  water  in  the  collecting  bottle  should  be  shaken,  to  distribute  the 
bacteria  which  may  have  settled  to  the  bottom,  before  drawing  off  by 
means  of  a  sterilized  pipette  the  amount  used  for  the  experiment,  and 
the  germs  present  in  it  are  to  be  distributed  through  the  liquefied 
gelatin  by  gently  moving  the  tube  to  and  fro. 

Koch's  method  of  preparing  a  gelatin  plate  is  illustrated  in  Fig. 
1'JO.  A  glass  dish,  containing  ice  water  and  covered  with  a  large 


.  lit*;. 


plate  of  glass,  is  supported  upon  a  levelling  tripod.  By  means  of  a 
spirit  level  this  is  adjusted  to  a  horizontal  position,  so  that  when  the 
liquefied  gelatin  is  poured  upon  the  smaller  sterilized  glass  plate,  seen 
in  the  centre  of  the  large  plate  of  glass,  it  will  not  flow,  but  may  be 
evenly  distributed  over  the  surface  by  means  of  a  sterilized  glass  rod. 
The  glass  cover  resting  against  the  side  of  the  apparatus  is  placed 
over  the  gelatin  plate  while  it  is  cooling,  to  protect  it  from  atmo- 
spheric germs,  and  when  the  gelatin  is  hard  the  plate  is  transferred 
to  a  shallow  glass  dish,  which  is  kept  at  a  temperature  of  about 
20°  C.  for  several  days  for  the  development  of  colonies.  It  is  difficult 
to  count  colonies  when  more  than  five  thousand  develop  upon  a  plate 
of  the  usual  size,  and  for  this  reason  it  will  be  best  to  repeat  the  ex- 
periment with  a  smaller  quantity  of  water  from  the  same  source,  if 
this  is  at  hand,  rather  than  to  attempt  to  count  an  overcrowded 
plate.  Before  pouring  the  gelatin  upon  the  plate  the  lip  of  the  test 
tube  containing  it  should  be  sterilized  by  passing  it  through  a  flame. 
The  liquefied  gelatin  should  ba  carefully  distributed  to  cover  a  rect- 


630  BACTERIA   IN   WATER. 

angular  surface  and  leaving  a  margin  of  about  one  centimetre  around 
the  edge  of  the  plate.  The  Koch's  dish  in  which  the  gelatin  plate  is 
placed  for  the  development  of  colonies  should  be  carefully  sterilized 
by  heat  or  by  washing  it  out  with  a  sublimate  solution.  A  circular 
piece  of  filtering  paper,  saturated  with  sublimate  solution  or  distilled 
water,  is  placed  at  the  bottom  of  the  lower  dish  to  keep  the  air  in  a 
moist  condition  and  prevent  drying  of  the  gelatin.  Usually  two  or 
three  plates  made  at  the  same  time  are  placed  one  above  the  other  on 
glass  supports  made  for  this  purpose.  If  many  liquefying  organisms 
are  present  it  will  be  necessary  to  count  the  colonies  before  these  run 
together — usually  on  the  second  day  ;  but  in  the  absence  of  liquefy- 
ing colonies  it  is  best  to  wait  until  the  third,  or  even  the  fifth  day,  as 
the  number  of  visible  colonies  and  the  ease  of  counting  them  will  be 
greater  than  at  an  earlier  date.  The  development  of  a  few  scattered 
liquefying  colonies  which  threaten  to  spoil  the  plate  may  be  arrested 
by  taking  up  the  liquefied  gelatin  from  each  with  a  bit  of  filtering 
paper,  and  then,  by  means  of  a  camers-hair  brush,  applying  a  solu- 
tion of  potassium  permanganate  to  the  margin  of  the  colony.  The 
growth  of  colonies  of  mould  fungi,  which  have  developed  from  spores, 
from  the  atmosphere  falling  upon  the  plate  while  it  is  exposed,  can 
be  checked  by  the  application  of  collodion  containing  bichloride  of 
mercury. 

Counting  of  the  colonies  is  a  simple  matter  when  they  are  few 
in  number ;  when  they  are  numerous  it  is  customary  to  place  the 
plate  over  a  dark  background,  and  to  place  above  it  a  glass  plate 
divided  into  square  centimetres  by  lines  ruled  with  a  diamond.  By 
means  of  a  lens  of  low  power  the  colonies  in  a  certain  number  of 
squares  are  counted  and  the  average  taken.  This  multiplied  by  the 
number  of  square  centimetres  in  the  gelatin-covered  surface  gives 
approximately  the  entire  number  of  colonies  which  have  developed 
from  the  amount  of  water  used  in  the  experiment. 

Instead  of  using  Koch's  original  plate  method,  as  above  described, 
the  shallow,  covered  glass  dishes  recommended  by  Petri  may  bo 
employed.  These  are  from  one  to  one  and  one-half  centimetres  high 
and  from  ten  to  fifteen  centimetres  in  diameter.  The  liquefied  gel- 
atin is  poured  into  the  lower  dish  and  the  cover  at  once  placed  over 
it.  The  gelatin  does  not  dry  out  very  soon,  but,  if  necessary,  several 
of  these  Petri's  dishes  may  be  placed  in  a  larger  jar,  which  serves  as 
a  moist  chamber. 

The  roll  tubes  of  Esmarch  may  also  be  used,  and  have  the  ad- 
vantage that  accidental  colonies  from  air-borne  germs  are  excluded. 
The  counting  of  colonies  is  not  quite  as  easy,  but  by  the  use  of  a 
mounted  lens  especially  designed  for  the  purpose  it  is  attended  with 
no  great  difficulty.  The  surface  of  the  tube  is  divided  into  squares 


BACTERIA    IX   WATER.  631 

by  colored  lines,  and  the  number  of  colonies  in  several  squares  is 
counted  in  order  to  obtain  an  average  and  estimate  the  entire 
number. 

Water  which  contains  numerous  liquefying  bacteria  had  better 
be  examined  by  the  use  of  nutrient  agar  instead  of  gelatin;  and  in 
very  warm  weather  it  will  be  necessary  to  use  an  agar  medium,  as 
ten-per-cent  gelatin  is  likely  to  melt  if  the  temperature  goes  above 
22°  C.  A  difficulty  in  the  use  of  agar  for  plates  consists  in  the  lia- 
bility of  the  film  to  slip  from  the  glass.  This  may  be  remedied  to 
some  extent  by  adding  a  few  drops  of  a  concentrated  solution  of  gum 
acacia  to  the  liquefied  agar  medium.  Petri's  dishes  are  well  adapted 
for  the  use  of  the  agar  medium,  as  the  objection  referred  to  does  not 
apply  to  them.  The  gelatin-agar  medium,  containing  5  per  cent 
of  gelatin  and  0. 75  per  cent  of  agar,  may  also  be  used  with  advan- 
tage in  the  bacteriological  analysis  of  water.  Much  stress  was  at 
one  time  laid  upon  the  enumeration  of  liquefying  colonies,  upon 
the  supposition  that  the  liquefying  bacteria  were  especially  harmful 
as  compared  with  the  non-liquefying,  and  that  a  water  containing 
many  liquefying  colonies  was  to  be  looked  upon  with  suspicion.  We 
now  know,  however,  that  there  are  many  common  and  harmless 
saprophytes  which  cause  the  liquefaction  of  gelatin,  and  that  some 
of  the  most  dangerous  pathogenic  bacteria  do  not  liquefy  gelatin. 
This  distinction  has  therefore  no  special  value,  and  the  question  for 
bacteriologists  to-day  is  not  how  large  is  the  comparative  number  of 
liquefying  colonies,  but  what  species  are  represented  by  the  colonies 
present,  liquafying  and  non-liquefying,  and  what  are  the  special 
pathogenic  properties  of  each.  The  answer  to  these  questions,  in 
the  case  of  any  particular  water  supply,  calls  for  special  knowledge 
and  great  patience  and  care  in  the  isolation  in  pure  cultures,  and 
careful  study  of  the  various  species  present. 

It  is  now  generally  recognized  that  a  mere  enumeration  of  the 
number  of  colonies  which  develop  from  a  water  under  investigation 
is  not  a  sufficient  indication  upon  which  to  found  an  opinion  as  to  its 
potability.  An  excessive  number  of  bacteria  is  an  indication  that 
the  water  contains  a  large  amount  of  the  organic  material  which 
serves  as  pabulum  for  these  microorganisms.  But  the  chemists  are 
able  to  determine  the  amount  of  organic  matter  present  in  water 
with  greater  precision  ;  and,  as  we  have  seen,  the  number  of  bacteria 
may  increase  many-fold  in  water  which  is  kept  standing  in  the  labo- 
ratory for  two  or  three  days  in  a  well-corked  bottle.  As  a  matter  of 
fact,  the  enumeration  of  bacteria  in  water,  although  it  has  given  us 
results  of  scientific  interest,  has  not  materially  added  to  the  methods 
previously  applied  for  estimating  the  sanitary  value  of  water  ob- 
tained from  various  sources  for  drinking  purposes.  But  the  bacte- 


632  BACTERIA    IX   AVATER. 

riological  examination  may  prove  to  be  of  great  value  if  it  succeeds 
in  demonstrating  the  presence  of  certain  pathogenic  bacteria  and  in 
thus  preventing  the  use  of  a  dangerous  water.  We  do  not  mean  to 
say,  however,  that  an  enumeration  of  the  bacteria  present  in  drink- 
ing water  has  no  practical  value.  An  excessive  number  indicates  an 
excessive  amount  of  organic  pabulum,  which  may  have  come  from 
a  dangerous  source;  and  the  dangerous  pathogenic  bacteria  are  not 
only  more  likely  to  be  present  in  such  water,  but  they  can  more 
readily  multiply  in  it,  while  in  a  pure  water  they  would  fail  to  in- 
crease in  number,  and,  as  has  been  shown  by  experiment,  would  die 
out  within  a  short  time. 

The  number  of  bacteria  present  in  rain  water,  or  in  snow  which 
has  recently  fallen,  varies  greatly  at  different  times.  Naturally  the 
number  is  greater  when  the  surface  of  the  earth  is  dry  and  the  at- 
mosphere loaded  with  dust  by  currents  of  wind  passing  over  it,  and 
less  when  the  surface  is  moist  and  the  atmosphere  has  been  purified 
by  recent  rains. 

In  snow  from  the  surface  of  a  glacier  in  Norway,  Schmelck  found 
two  bacteria  and  two  spores  of  mould  fungi  per  cubic  centimetre  of 
water  from  the  melted  snow.  Ganowski,  in  experiments  made  with 
freshly  fallen  snow  collected  in  the  vicinity  of  Kiew,  obtained  the  fol- 
lowing results  :  February  2d,  1888  :  temperature  of  the  air,  7.2°  C. ; 
snowfall,  0. 1  millimetre ;  number  of  bacteria  in  1  cubic  centimetre 
of  water  from  melted  snow,  34  in  one  sample  and  38  in  another. 
February  20th,  1888:  temperature,  11.1°  C.  ;  snowfall,  1.1  milli- 
metres ;  number  of  bacteria  in  one  sample,  203,  in  another  384. 

Miquel  obtained  from  rain  water  collected  at  Montsouri  during  a 
rainy  season  4. 3  germs  per  cubic  centimetre  ;  in  rain  water  collected 
in  the  centre  of  the  city  of  Paris,  19  per  cubic  centimetre. 

.HcuZhas  also  been  shown  to  contain  bacteria  inconsiderable  num- 
bers. Bujwid  found  in  hailstones  which  fell  at  Warsaw  21,000 
bacteria  in  1  cubic  centimetre  ;  but  this  is  exceptional,  and  is  supposed 
to  be  due  to  the  fact  that  surface  water  had  been  carried  into  the  air 
by  the  storm  and  frozen.  Fontin  examined  hail  which  fell  in  St. 
Petersburg,  and  obtained  an  average  of  729  bacteria  per  cubic  centi- 
metre of  water  from  the  melted  hail. 

River  water  has  been  carefully  examined  by  numerous  bacterio- 
logists in  various  localities  and  at  different  seasons  of  the  year.  We 
give  below  some  of  the  results  reported  : 

Water  of  the  Seine  at  Choisy,  before  reaching  Paris,  300  ;  at 
Bercy,  1,200 ;  at  Saint-Denis,  after  receiving  the  sewer  water  from 
the  city,  200,000  germs  per  cubic  centimetre  (Miquel). 

Water  of  the  Spree  beyond  Kopenick,  82,000  ;  two  hundred  steps 
below  the  mouth  of  the  Wuhle,  118,000  ;  in  Berlin  above  the  mouth 


BACTERIA   IX   WATER.  633 

of  the  Panke,    040.000;  below  the  mouth  of  the  Panke,   1,800,000 
(Koch). 

Water  of  the  Main  above  the  city  of  Wiirzburg,  in  the  month  of 
February,  530  ;  below  the  city,  15,500  (Rosenberg). 

Water  of  the  Potomac,  at  Washington,  in  1886  :  January,  3,774  ; 
February,  2,536;  March,  1,210;  April,  1,521;  May,  1,004;  June, 
348  ;  July,  255  ;  August,  254 ;  September,  178 ;  October,  75  ;  No- 
vember, 116  ;  December,  967  (Theobald  Smith). 

The  Thames,  in  the  autumn  of  1885,  in  the  vicinity  of  London 
Bridge  two  hours  after  high  water,  contained  45,000  germs  per  cubic 
centimetre ;  the  water  of  the  Lea  at  Lea  Bridge,  4,200,000  (Bisch- 
off). 

The  Neva  inside  the  city  of  St.  Petersburg,  in  September,  1883, 
contained  1,500  in  one  sample  and  1,040  in  another  ;  in  November 
(20th),  6,500  (Poehl). 

The  water  of  the  Oder,  collected  within  the  limits  of  the  city  of 
Stettin,  was  found  by  Link  to  contain  from  5,240  to  15,000  bacteria 
per  cubic  centimetre  ;  that  of  the  Limmat,  at  Zurich,  346  in  one 
specimen  and  508  in  another  (Cramer). 

Lake  water,  as  a  rule,  contains  fewer  bacteria  than  river  water. 

Wolff htigel,  in  researches  extending  from  July,  1884,  to  July, 
1885,  obtained  from  the  water  of  the  Tegeler  Lake  an  average  of  396 
bacteria  per  cubic  centimetre.  Cramer  obtained  an.  average  of  168 
per  cubic  centimetre  during  the  months  of  October,  December,  and 
January,  1884,  from  the  water  of  Lake  Zurich  ;  in  June  of  the  same 
year  the  average  of  42  examinations  gave  71  per  cubic  centimetre. 
In  'Lake  Geneva,  Fol  and  Dunant  obtained  from  water  collected  some 
distance  from  the  shore  an  average  of  38  bacteria  per  cubic  centi- 
metre. 

Ice  which  is  usually  collected  from  lakes  and  rivers  contains  a 
greater  or  less  number  of  bacteria,  according  to  the  depth  and  purity 
of  the  water.  The  ice  used  in  Berlin,  collected  from  the  surface  of 
lakes  and  rivers  in  the  vicinity  of  the  city,  contains  from  a  few  hun- 
dred to  25,000  bacteria  to  the  cubic  centimetre  (Friinkel).  In  the  ex- 
periments of  Heyroth  samples  of  ice  from  the  same  source  gave  less 
than  100  per  cubic  centimetre  in  three,  from  100  to  500  in  eight,  from 
500  to  1,000  in  six,  from  1,000  to  5,000  in  seven,  and  14,400  in  one. 

Prudden  obtained  from  Hudson  River  ice,  put  up  six  miles  below 
the  city  of  Albany,  an  average  of  398  bacteria  per  cubic  centimetre 
from  transparent  ice,  and  in  the  superficial  "snow  ice"  9,187.  Ice 
collected  lower  down  the  river  contained  an  average  of  189  in  the 
transparent  and  3,693  in  the  snow  ice. 

Ice  from  the  Dora  at  Turin  was  found  by  Bordoni-Uffreduzzi  to 
contain  from  120  to  3,546  bacteria  per  cubic  centimetre. 


634  BACTERIA    IX   WATER. 

Hydrant  ivater,  as  supplied  to  cities,  has  received  the  attention 
of  numerous  investigators.  The  water  supply  of  Berlin  was  ex- 
amined by  Plagge  and  Proskauer  at  intervals  of  a  week  from  June, 
1885,  to  April,  1886.  Their  tabulated  results  show  considerable 
variations.  We  give  the  figures  for  a  single  day,  June  30th,  1885  : 
Stralauer  works,  water  of  the  Spree,  unfiltered  4,400,  filtered  53  ; 
Tegeler  works,  water  of  the  lake,  unfiltered  880,  filtered  44  ;  high  re- 
servoir at  Charlottenberg,  71  ;  75  W.  Wilhelmstrasse,  121  ;  Fried- 
richstrasse,  41-42  S.  W.,  160  ;  Schmidstrasse,  165  E.,  51  ;  Friedrich- 
strasse,  126  N.,  151  ;  Weinmeisterstrasse,  15  C.,  63. 

Wells  which  are  supplied  by  water  from  deep  strata  contain  few 
bacteria,  unless  contaminated  by  surface  water  in  which  they  are 
usually  very  abundant.  Roth  examined  the  water  of  sixteen  surface 
wells  in  Belgard,  which  has  a  very  porous  subsoil,  and  found  from 
4,500  to  5,000  bacteria  in  three,  from  7,800  to  15,000  in  six,  from 
18,000  to  35,000  in  six,  and  130,000  per  cubic  centimetre  in  one. 

Forty-seven  wells  in  Stettin,  the  water  of  which  was  examined  by 
Link,  gave  the  following  results  :  Less  than  100  in  six,  100  to  500  in 
twenty-one,  and  in  the  remainder  (sixteen)  from  1,000  to  18,000. 

Sixty-four  wells  in  Mainz  examined  by  Egger,  and  53  in  Gotha 
by  Becker,  gave  more  favorable  results  ;  the  number  of  wells  in  the 
former  city,  in  which  less  than  100  colonies  developed  from  1  cubic 
centimetre,  was  34,  and  in  the  latter  the  same  (34).  Bolton  examined 
the  water  of  13  wells  in  Gottingen,  and  found  but  1  in  which  the 
number  of  colonies  from  1  cubic  centimetre  was  less  than  100  ;  in  12 
the  number  varied  from  180  to  4,940. 

The  water  of  deep  wells  and  springs  may  be  entirely  free  from 
bacteria,  or  nearly  so.  Egger  found  in  the  water  of  an  artesian  well 
at  Mainz  4  bacteria  per  cubic  centimetre,  and  the  same  number  was 
found  by  Hueppe  in  the  deep  well  at  the  Wiesbaden  slaughter-house. 
The  artesian  well  at  the  gasworks  of  Kiel  was  found  by  Brennig  to 
contain  from  6  to  30  bacteria  per  cubic  centimetre.  In  a  spring  at 
Batiolettes,  Fol  and  Dunant  found  57  bacteria  per  cubic  centimetre. 
Fiirbringer  obtained  from  springs  at  Jena  156  from  one,  51  from 
another,  32  from  another,  and  109  from  another.  The  water  supplied 
to  Danzig  from  the  Prangenaur  Spring  was  found  in  several  experi- 
ments to  be  free  from  bacteria  (Freimuth). 

In  a  summary  of  results  obtained  in  various  German  cities  Tie  • 
mann  and  Gartner  find  that  sixty-nine  per  cent  of  the  wells  from 
which  samples  of  water  were  examined  contained  less  than  500  bac- 
teria per  cubic  centimetre. 

The  water  of  seivers  is  naturally  rich  in  bacteria.  Miquel  found 
that  at  Clichy  the  sewer  water  contained  6,000,000  bacteria  per  cubic 
centimetre.  Bischoff  found  in  water  from  London  sewers  7,500,000, 


BACTERIA    IX   WATER.  635 

and  numerous  observations  show  that  tho  number  of  bacteria  in  river 
water  is  greatly  increased  in  tlia  vicinity  of  and  below  the  mouths 
of  city  sewers. 

We  conclude  from  the  experimental  data  recorded  that  water 
containing  less  than  100  bacteria  to  the  cubic  centimetre  is  presum- 
ably from  a  deep  source  and  uncontaminated  by  surface  drainage, 
and  that  it  will  usually  be  safe  to  recommend  such  water  for  drink- 
ing purposes,  unless  it  contains  injurious  mineral  substances. 
Water  that  contains  more  than  500  bacteria  to  the  cubic  centimetre, 
although  it  may  in  many  cases  be  harmless,  is  to  be  looked  upon 
with  some  suspicion,  and  water  containing  1,000  or  more  bacteria  is 
presumably  contaminated  by  sewage  or  surface  drainage  and  should 
be  rejected  or  filtered  before  it  is  used  for  drinking  purposes.  But, 
as  heretofore  .stated,  the  danger  does  not  depend  directly  upon  the 
number  of  bacteria  present,  but  upon  contamination  with  pathogenic 
species  which  are  liable  to  be  present  in  surface  water  and  sewage. 
In  swallowing  a  glassful  of  pure  spring  water  a  number  of  bacteria 
from  the  buccal  cavity  are  washed  away  and  carried  into  the  stomach, 
which,  if  enumerated,  would  doubtless  far  exceed  in  numbers  those 
found  in  the  most  impure  river  water. 

The  number  of  bacteria  does  not  depend  alone  upon  the  amount 
of  organic  pabulum  contained  in  a  water,  and  cannot  be  depended 
upon  in  forming  an  estimate  of  this ;  for,  as  has  been  shown  by 
Bolton,  certain  water  bacteria  multiply  abundantly  in  water  con- 
taining comparatively  little  organic  matter,  while  other  species  fail 
to  grow  unless  the  quantity  is  greater.  In  a  water  containing  con- 
siderable nutrient  material  the  water  bacteria  may  be  restrained  in 
their  development  by  other  species  present  until  the  amount  of  pabu- 
lum is  reduced  so  that  these  no  longer  thrive,  when  the  common 
water  bacteria  will  take  the  precedence,  and  an  enumeration  may 
show  a  greater  number  of  colonies  than  at  first.  But,  in  general, 
water  rich  in  organic  material  contains  a  greater  number  of  bacteria 
and  a  greater  variety  of  species  than  that  which  is  comparatively 
pure. 

That  certain  bacteria  may  multiply  in  water  which  has  been 
carefully  distilled  has  been  shown  by  Bolton  and  others.  Two  com- 
mon water  bacteria — Micrococcus  aquatilis  and  Bacillus  erythrospo- 
rus — multiplied  abundantly  in  doubly  distilled  water,  and  when 
this  water  was  again  sterilized  and  re-inoculated  with  one  of  these 
species  the  same  abundant  increase  occurred.  This  was  repeated  six 
times  with  the  same  result  (Bolton).  Computing  the  number  of 
these  water  bacteria  in  ten  cubic  centimetres  of  distilled  water  at 
twenty  millions,  and  estimating  their  specific  gravity  at  one,  and  the 
diameter  of  the  individual  cells  at  one  yw,  the  total  weight  of  the  entire 


BACTERIA   IN   WATER. 

number,  according  to  Bolton,  would  be  less  than  one-hundredth 
of  a  milligramme,  and  at  least  three-fourths  of  this  must  consist  of 
water.  The  organic  material  represented  by  this  number  of  bacteria 
would  therefore  be  so  minute  that  it  might  be  supplied  by  dust  par- 
ticles accidentally  falling  into  the  distilled  water. 

Rosenberg  has  shown  that  while  many  of  the  species  which  he 
obtained  in  pure  cultures  from  the  water  of  the  river  Main  multiplied 
in  sterilized  distilled  water,  other  species  quickly  died  out  in  such 
water.  The  growth  of  certain  bacteria  depends  not  only  upon  the 
quantity  of  nutritive  material  present,  but  upon  its  quality,  the  con- 
ditions in  this  regard  being  widely  different  for  different  species.  . 

In  view  of  the  facts  heretofore  stated  bacteriologists  are  now  giv- 
ing more  attention  to  a  careful  study  of  the  kinds  of  bacteria  pre- 
sent in  their  examinations  of  water.  Rosenberg,  in  his  examinations 
of  the  water  of  the  Main  in  the  vicinity  of  Wiirzburg  (1886),  found 
that  before  the  river  reached  the  city  the  water  contained  more 
micrococci  than  bacilli,  but  that  after  receiving  the  sewage  of  the 
city  the  number  of  bacilli  was  greatly  in  excess. 

Adametz  (1888)  has  described  eighty-seven  species  obtained  by 
him  from  water  in  the  vicinity  of  Vienna  ;  Maschek  found  fifty-five 
different  species  in  the  drinking  water  used  at  Laitmeritz;  and  Tils 
(1890)  has  described  fifty-nine  species  obtained  by  him  from  the  city 
water  supply  at  Freiburg. 

Among  the  pathogenic  bacteria  which  are  liable  to  find  their 
way  into  water  used  for  drinking  purposes,  the  most  important,  from 
a  sanitary  point  of  view,  are  the  bacillus  of  typhoid  fever  and  the 
spirillum  of  Asiatic  cholera.  Both  of  these  microorganisms  are  pre- 
sent in  great  numbers  in  the  excreta  of  persons  suffering  from  the 
specific  forms  of  disease  to  which  they  give  rise,  and  are  consequently 
liable  to  contaminate  wells  and  streams  which  receive  surface  water, 
when  such  excreta  are  thrown  upon  the  surface  or  into  sewers,  etc. 
Epidemics  of  these  diseases  have  frequently  been  traced  to  the  use 
of  such  contaminated  water,  and  in  a  few  instances  the  presence  of 
these  specific  disease  germs  in  water  has  been  demonstrated  by  bac- 
teriological methods.  Laboratory  experiments  indicate,  however, 
that  an  increase  of  these  pathogenic  bacteria  in  drinking  water  is  not 
likely  to  occur,  except  under  special  conditions,  and  that  they  die 
out  after  a  time,  being  at  a  disadvantage  in  the  struggle  for  exist- 
ence constantly  going  on  among  the  numerous  species  which  have 
their  normal  habitat  in  water. 

Bolton,  Frankland,  and  others  have  shown  that  the  anthrax  ba- 
cillus, not  containing  spores,  dies  out  in  hydrant  water  within  five  or 
six  days.  In  the  experiments  of  Kraus  the  anthrax  bacillus  added 
to  well  water,  not  sterilized,  at  a  temperature  of  10.5°  C.,  was  still 


BACTERIA   IN   WATER.  637 

present  in  a  living  condition  on  the  second  day,  but  110  colonies  de- 
veloped aftar  the  third  day ;  the  typhoid  bacillus  died  out  between 
the  fifth  and  seventh  days  ;  the  cholera  spirillum  was  no  longer  found 
on  the  second  day.  In  the  meantime  the  common  water  bacteria 
had  increased  in  numbers  enormously.  Similar  results  have  been 
reported  by  Hochstetter  and  others.  Hueppe,  in  ten  experiments  in 
which  the  typhoid  bacillus  was  added  to  well  water  of  a  bad  quality, 
found  that  in  two  no  development  of  this  bacillus  occurred  after  the 
fifth  day,  while  a  few  colonies  developed  in  the  other  experiments  as 
late  as  the  tenth  day.  In  these  experiments  the  temperature  was 
comparatively  low  (10.5°  C.).  At  a  higher  temperature  the  experi- 
ments of  Wolffhugel  and  Riedel  show  that  an  increase  may  take 
place.  At  the  room  temperature  (about  20°  C.)  the  typhoid  bacillus 
added  to  distilled  water,  to  well  water,  and  to  Berlin  hydrant  water 
was  still  present,  in  some  instances,  at  the  end  of  thirty-two  days. 
And  it  was  found  that  in  some  cases  a  decrease  in  the  number 
occurred,  then  a  notable  increase,  and  finally  a  second  diminution. 

Koch  found  the  cholera  spirillum  in  a  water  tank  at  Calcutta 
during  a  period  of  fourteen  days,  and  in  his  experiments  showed  that 
it  preserved  its  vitality  in  well  water  for  thirty  days,  in  Berlin  sewer 
water  for  six  to  seven  days,  and  in  the  same  mixed  with  faeces  for 
twenty-seven  hours  only.  In  the  experiments  of  Nicati  and  Rietsch 
the  cholera  spirillum  preserved  its  vitality  in  distilled  water  for 
twenty  days,  in  sewer  water  (of  Marseilles)  thirty-eight  days,  in 
water  of  the  harbor  for  eighty-one  days.  The  numerous  experiments 
recorded  by  the  observers  named,  and  by  Bolton,  Hueppe,  Hoch- 
stetter, Maschek,  Kraus,  and  others,  show  that  while  the  cholera 
spirillum  may  sometimes  quickly  die  out  in  distilled  water,  in  other 
experiments  it  preserves  its  vitality  for  several  weeks  (Maschek),  and 
that  it  lives  still  longer  in  water  of  bad  quality,  such  as  is  found  in 
sewers,  harbors,  etc.  Bolton  found  that  for  its  multiplication  a 
water  should  contain  at  least  40  parts  in  100,000  of  organic  material, 
while  the  typhoid  bacillus  grew  when  the  proportion  was  considerably 
less  than  this— 6.7  parts  in  100,000. 

Russell  (1891)  has  studied  the  bacterial  flora  of  the  Gulf  of 
Naples,  and  of  the  mud  at  the  bottom  of  this  gulf,  collected  at 
various  depths  up  to  eleven  hundred  metres.  His  investigations 
show  that  sea  water  does  not  contain  as  many  bacteria  as  an 
equal  volume  of  fresh  water;  that  bacteria  are  found  in  about 
equal  numbers  in  water  from  the  surface  and  in  that  from  various 
depths  ;  that  the  mud  at  the  bottom  constantly  contains  large  num- 
bers of  bacteria ;  that  some  of  the  species  isolated  grow  best  in  a 
Culture  medium  containing  sea  water. 

At  a  depth  of  50  metres  the  water  contained  121  bacteria  per  cubic 


BACTERIA    IX    WATER. 

centimetre,  and  the  mud  from  the  bottom  245,000  ;  at  100  metres  the 
water  contained  10  and  the  mud  200,000  per  cubic  centimetre  ;  at 
500  metres  the  water  contained  22  and  the  mud  12,500  per  cubic 
centimetre  ;  at  1,100  metres  the  mud  contained  24,000. 

The  following  new  species  were  obtained  by  Russell  from  the 
source  mentioned  :  Bacillus  thalassophilus,  Cladothrix  intricata, 
Bacillus  granulosus,  Bacillus  limosus,  Spirillum  marinum,  Bacillus 
litoralis, 'Bacillus  halophilus. 

The  bacterial  flora  of  fresh  and  sea  water  is  very  extensive,  as 
will  be  seen  by  the  following  list  of  species  which  have  been  described 
by  various  bacteriologists  who  have  given  their  attention  to  its 
study  : 

XOX-PATHOGEXIC    MICROCOCCI. 

Micrococcus  aurantiacus  (Cohii),  Micrococcus  luteus  (Cohn),  Micrococcus 
violaceus  (Cohn),  Micrococcus  flavus  liquefaciens  (Fliigge),  Micrococcus  fla- 
vus  desideiis  (Fliigge),  Micrococcus  radiatus  (Fliigge),  Micrococcus  ciimaba- 
reus  (Fliigge),  Micrococcus  flavus  tardigradus  (Fliigge),  Micrococcus  versi- 
color  (Fliigge),  Micrococcus  agilis  (Ali-Cohen),  Micrococcus  fuscus  (Maschek), 
Diplococcus  luteus  (Adametz),  Pediococcus  albus  (Lindner),  Micrococcus 
cerasiiius  siccus  (List),  Micrococcus  citreus  (List),  Micrococcus  aquatilis 
(Bolton),  Micrococcus  fervidosus  (Adametz),  Micrococcus  plumosus  (Brauti- 
gam),  Micrococcus  viticulosus  (Katz),  Micrococcus  cremoides(Zimmermann), 
Micrococcus  carneus  (Zimmerman.n),  Micrococcus  concentricus  (Zimmer- 
mann),  Micrococcus  rosettaceus  (Zimmermann),  Micrococcus urese  (Pasteur), 
Weisser  Streptococcus  (Maschek),  Wurmformiger  Streptococcus  (Maschek), 
Micrococcus  aerogenes  (Miller),  Sarcina  alba,  Sarcina  Candida  (Reinke), 
Sarcina  lutea. 

PATHOGENIC   MICROCOCCI. 

Staphylococcus  pyogenes  aureus  (Rosenbach),  Micrococcus  of  Heyden- 
reich — "  Micrococcus  Biskra/' 

XOX-PATHOGEXIC   BACILLI. 

Bacillus  arborescens  (Fraiikland),  Bacillus  viscosus  (Frankland),  Bacil- 
lus aquatilis  (Frankland),  Bacillus  liquidus  (Frankland),  Bacillus  nubilis 
(Frankland),  Bacillus  vermicularis  (Frankland),  Bacillus  aurantiacus 
(Frankland),  Bacillus  coeruleus  (Smith),  Bacillus  glaucus  (Maschek),  Bacil- 
lus albus  putidus  (Maschek),  Bacillus  fluoresceiis  liquefaciens.  Bacillus  ftuo- 
rescens  mvalis  (Schmolck),  Bacillus  lividus  (Plagge  and  Prpskauer),  Bacil- 
lus rubidus  (Eisenberg),  Bacillus  sulfureum  (Holschewriikoff),  Bacillus 
violaceus,  Bacillus  gasoformans  (Eisenberg),  Bacillus  liquefaciens  (Eisen- 
berg), Bacillus  phosphorescens  indicus  (Fischer),  Bacillus  phosphoresces s 
indigenus  (Fischer),  Bacillus  phosphorescens  gelidus  (Katz),  Bacillus  sma- 
ragdino-phosphoresceiis  (Katz),  Bacillus  argenteo-pbosphorescens  Nos.  I., 
II.,  and  III.  (Katz),  Bacillus  cyaneo-phosphoresceiis  (Katz),  Bacillus  ar- 
genteo-phosphorescens  liquefaciens  (Katz),  Bacillus  ramosus,  Bacillus  sub- 
tilis  (Ehrenberg),  Proteus  sulfureus  (Lindenborn),  Bacillus  aureus  (Ada- 
metz), Bacillus  brunneus  (Adametz),  Bacillus  flavocoriaceus  (Adametz), 
Bacillus  fluorescensnon-liquefaciens,  Bacillus  latericeus  (Adametz),  Bacillus 
stolonatus  (Adametz),  Bacillus  berolinensis  indicus  (Classen),  Bacillus  ery- 
throsporus  (Eidam),  Bacillus  luteus  (List),  Bacillus  aquatilis  sulcatus  Nos. 
1,  2,  3,  4,  ar>d  5  (Weichselbaum),  Bacillus  albus  (Eisenberg),  Bacillus  multi- 
pediculosus( Fliigge),  Bacillus  Ziirnianum  (List),  Bacillus  fulvus  (Zimmer- 
mann), Bacillus  helvolus  (Zimmermann),  Bacillus  ochraceus  (Zimmer- 


BACTERIA   IN   WATER.  639 

maim),  Bacillus plicatus,  Bacillus  devorans  (Zimmermann),  Bacillus  gracilis 
(Zimmermann),  Bacillus  guttatus  (Zimmermann),  Bacillus  implexus  (Zim- 
mermann), Bacillus  punctatus  (Zimmermann),  Bacillus  radiatus  aquatilis 
(Zimmermann),  Bacillus  vermiculosus  (Zimmermann),  Bacillus  constrictus 
(Zimmermann),  Bacillus  fluorescens  aureus  (Zimmermann),  Bacillus  fluo- 
rescens  longus  (Zimmermann),  Bacillus  fluorescens  tenuis  (Zimmermann), 
Bacillus  fuscus  (Zimmermann),  Bacillus  rubefaciens  (Zimmermann),  Bacil- 
lus subflavus  (Zimmermann),  Bacillus  janthinus  (Zopf),  Bacillus  mycoides 
(Fliigge),  Bacillus  tremelloides  (Tils),  Bacillus  cuticularis  (Tils),  Bacillus 
filiformis  (Tils),  Bacillus  ubiquitus  (Jordan),  Bacillus  circulans  (Jordan), 
Bacillus  superficialis  (Jordan),  Bacillus  reticularis  (Jordan),  Bacillus  ru- 
bescens  (Jordan),  Bacillus  hyalinus  (Jordan),  Bacillus  cloacae  (Jordan), 
Bacillus  delicatulus  (Jordan),  Bacillus  violaceus  laurentius  (Jordan). 

PATHOGENIC  BACILLI. 

Bacillus  typhi  abdominalis  (Eberth,  Gaffky),  Bacillus  erysipelatos  suis 
("  Bacillus  murisepticus, "  Koch),  Bacillus  septicaemias  haemorrhagicae 
("Bacillus  cuniculicida,"  Koch),  Proteus  vulgaris  (Hauser),  Proteus  mira- 
bilis  (Hauser),  Bacillus  canalis  capsulatus  (Mori),  Bacillus  canal  is  parvus 
(Mori),  Spirillum  choleroe  Asiaticse  (''Comma  bacillus,"  Koch),  Bacillus  coli 
communis  (Escherich),  Bacillus  hydrophilus  fuscus  (Sanarelli),  Bacillus 
venenosus  (Vaughan),  Bacillus  yenenosus  brevis  (Vaughan),  Bacillus  vene- 
nosus  invisibilis  (Vaughan),  Bacillus  venenosus  liquefaciens  (Vaughan). 

The  following  additional  species  are  described  by  Zimmermann  (1894)  in 
his  second  publication  ("Die  Bakterien  unserer  Trink-  und  Nutzwasser "). 
Micrococcus  candidus,  Micrococcus  coralloides,  Streptococcus  cinereus,  Mi- 
crococcus  sulphureus,  Micrococcus  galbaiiatus,  Micrococcus  erythromyxa, 
Sarcina  flavea,  Sarcina  aurantiaca,  Sarcina  rosea.  Bacillus  ruber,  Bacillus 
miniaceus,  Bacillus  mesentericus  roseus,  Bacillus  carnosus,  Bacillus  chryso- 
gloia,  Bacillus  multipediculus  flavus,  Bacillus  villosus,  Bacillus  radiatus, 
Bacillus  fluorescens  albus,  Bacillus  viridans,  Bacillus  turcosa,  Bacillus  halans, 
Bacillus  nacreaceus,  Bacillus  mirabilis,  Bacillus  umbilicatus,  Bacillus  lactis 
viscosus,  Bacillus  synxaiithus,  Bacillus  sericeus,  Bacillus  minutus,  Bacillus 
stellatus,  Bacillus  radicosus,  Bacillus  vernicosus,  Bacillus  mucosus,  Bacillus 
centralis,  Bacillus  spumosus,  Bacillus  aimulatus,  Bacillus  liquefaciens,  Bacil- 
lus disciformans. 

The  following  spirilla  and  "  vibrios"  have  also  been  found  in  water — 
chiefly  in  river  water  : 

Spirillum  volutans,  Spirillum  sanguineum,  Spirillum  serpens,  Vibrio  ru- 
gula,  Spirillum  plicatile,  Spirillum  marinum  (Russell).  Spirillum  cholerae 
Asiaticae,  Spirillum  of  Renon,  Vibrio  aquatilis  (Gunther),  Vibrio  of  Weibel, 
Vibrios  of  Bujwid  (Bacillus  choleroides  a  and  6),  Vibrio  of  Loftier,  Vibrios 
of  Bonhoff,  Vibrio  of  Blackstein,  Vibrios  of  Sanarelli,  Vibrios  of  Fischer, 
Vibrio  Berolinensis,  Vibrio  Danubicus,  Vibrio  of  Pfuhl  (v.  Metchnikovi  ?). 
Several  of  the  "  vibrios"  in  this  list  which  have  recently  been  obtained  from 
river  water  in  various  parts  of  Europe  are  probably  varieties  of  the  cholera 
spirillum. 

ADDITIONAL   NOTES    UPON    BACTERIA   IN   WATER. 

It  is  now  generally  recognized  by  bacteriologists  that  the  potability  of 
water  is  to  be  determined  by  an  investigation  relating  to  the  presence  or  ab- 
sence of  known  pathogenic  bacteria,  rather  than  by  an  estimate  of  the  num- 
ber of  bacteria  present  in  each  cubic  centimetre  of  the  water  under  exami- 
nation. From  a  sanitary  point  of  view  the  most  important  of  these  pathogenic 
bacteria  are  the  cholera  spirillum  and  allied  "  vibrios,"  the  bacilli  of  the  "  ty- 
phoid group "  (Bacillus  typhi  abdominalis  and  allied  forms),  the  bacilli  of 
the  "colon  group"  (Bacillus  coli  communis  with  its  varieties  and  similar 
bacilli  of  faecal  origin).  When  one  of  these  pathogenic  bacilli  is  present  in  a 


G40  BACTERIA   IN   WATER. 

water-supply  hi  small  numbers  as  compared  with  the  number  of  saprophytic 
bacteria,  it  is  not  an  easy  matter  to  demonstrate  the  fact  by  the  ordinary 
plate  method,  especially  in  the  case  of  non-liquefying  species  like  the  typhoid 
bacillus.  If  we  harve,  for  example,  one  typhoid  bacillus  to  one  thousand  ba- 
cilli of  other  species  it  is  evident  that  in  a  series  of  three  plates,  made  in  the 
usual  way  for  the  purpose  of  obtaining  isolated  colonies,  there  would  be  but  a 
small  chance  of  obtaining  a  colony  of  the  typhoid  bacillus  in  plate  No.  3, 
and  a  plate  containing  one  thousand  colonies  or  more  would  be  so  crowded 
that  the  detection  of  the  single  typhoid  colony  would  be  very  difficult.  For 
this  reason,  it  is  necessary  to  resort  to  special  methods  by  which  the  more 
numerous  saprophytic  bacteria  will  be  excluded,  or  their  numbers  greatly 
reduced.  Some  of  the  methods  which  have  been  successfully  employed  for 
the  detection  of  the  typhoid  bacillus  and  of  the  cholera  spirillum  are  given 
in  the  sections  devoted  to  these  microorganisms.  We  give  below  some  de- 
tails relating  to  the  methods  employed  by  bacteriologists  of  recognized  com- 
petence in  recent  investigations  : 

Marpmaim  (1895)  considers  all  water  which  contains  faecal  bacteria  as 
dangerous  as  a  supply  for  drinking  purposes.  For  the  detection  of  patho- 
genic bacteria  he  recommends  the  following  procedure : 

The  pathogenic  bacteria  are  divided  into  two  groups  by  cultivation  in  nu- 
trient agar  containing  0.2  percent  of  citric  acid,  and  in  the  same  medium  con- 
taining two  per  cent  of  sodium  carbonate.  The  bacilli  of  the  typhoid  group 
are  said  to  grow  in  the  acid  medium  but  not  in  that  containing  two  per  cent 
of  sodium  carbonate.  On  the  other  hand,  cholera  vibrios  develop  in  the  al- 
kaline medium  but  not  in  that  containing  0.2  per  cent  of  citric  acid.  The  ba- 
cilli of  the  colon  group  also  ("  cloaca-bacilli")  do  not  grow  in  the  medium 
containing  citric  acid.  Bouillon  containing  the  same  amounts  of  acid  and 
alkali  is  also  employed.  The  water  to  be  examined  is  first  mixed  with  an 
equal  portion  of  acid  and  of  alkaline  bouillon  in  two  test  tubes,  and  these  are 
kept  at  a  temperature  of  30°  C.  for  twenty -four  hours,  during  which  time 
the  pathogenic  bacteria,  if  present,  will  multiply  and  cause  a  clouding  of  the 
culture  media.  Inoculations  are  now  made  into  the  acid  arid  alkaline  agar 
and  gelatin.  Growth  in  alkaline  gelatin  at  the  room  temperature  (10°  to  18° 
C.)  is  due  to  "cloaca-bacteria"  ;  growth  in  acid  gelatin  at  20°  to  23°  C.  is  due 
to  bacilli  of  the  typhoid  group.  Plates  should  also  be  made  from  the  clouded 
bouillon,  acid  and  alkaline  ;  and  the  colonies  resembling  those  of  the  typhoid 
or  of  the  colon  group  should  be  tested  in  nutrient  gelatin  containing  sugar 
to  ascertain  whether  there  is  development  of  gas,  in  which  case  the  bacilli  are 
of  the  colon  group. 

When  typhoid  and  colon  bacilli  are  associated  in  water  the  last-mentioned 
bacillus  takes  the  precedence,  and  the  typhoid  bacillus  has  a  tendency  to  dis- 
appear. This  is  shown  by  the  experiments  of  Girnbert  (1894),  who  introduced, 
at  the  same  time,  colon  bacilli  and  typhoid  bacilli  into  water,  and  found  that 
at  the  end  of  forty-eight  hours  he  was  no  longer  able  to  isolate  the  typhoid 
bacillus  from  plates.  In  view  of  this  fact  failure  to  find  the  typhoid  bacillus 
does  not  relieve  the  water  from  the  suspicion  of  being  dangerous  if  the  colon 
bacillus  is  present.  But,  on  the  other  hand,  this  bacillus  is  so  common  that 
it  is  perhaps  the  exception  when  it  is  not  present  in  surface  waters.  As 
pointed  out  by  von  Freudenreich  (1895)  it  may,  however,  escape  detection 
unless  a  considerable  quantity  of  water  is  used  in  making  the  test.  When 
the  quantity  is  from  one  hundred  to  five  hundred  cubic  centimetres,  in- 
stead of  from  one  to  five  cubic  centimetres,  as  was  formerly  the  usual  amount 
employed,  it  is  found  not  infrequently  even  in  spring  water  (von  Freuden- 
reich). 

The  author  last  mentioned  says  that  when  present  in  small  numbers  it 
may  be  demonstrated  by  the  method  of  Vincent,  as  follows  :  Mix  of  the  water 
ninety  cubic  centimetres  with  ten  cubic  centimetres  of  a  twenty-per-cent 
solution  of  peptone,  and  one  cubic  centimetre  of  a  seven-per-cent  solution  of 
carbolic  acid;  place  in  the  incubating  oven  at  42  C.  If  development  «K> 


BACTERIA   IN   WATER.  641 

curs  it  will  probably  be  due  to  the  colon  bacillus,  but  it  will  be  necessary  to 
make  plates  and  pure  cultures  from  single  colonies  in  order  to  determine 
this  with  certainty.  The  demonstration  may  be  made  more  quickly,  accord- 
ing to  von  Freudeiireich,  by  using  a  medium  containing  milk  sugar  (five  per 
cent)  and  cultivating  at  35°  C.  If  the  colon  bacillus  is  present  there  will  be 
an  abundant  development  of  gas  in  from  twelve  to  twenty -four  hours,  and 
the  bacillus  may  then  be  readily  isolated  by  the  plate  method.  The  colon 
bacillus  has  been  found  byMoissan  and  Gimbert  in  mineral  waters  bottled  in 
France.  Poncet  (1895)  has  made  a  careful  study  of  the  bacteria  found  in  the 
various  springs  at  Vichy.  The  species  described  are  all  harmless  water  bac- 
teria and  have  little  interest  from  a  sanitary  point  of  view. 

Kruse  (1894),  as  a  result  of  his  extended  researches  and  of  a  critical  con- 
sideration of  the  experimental  data  available,  arrives  at  the  conclusion  that  a 
sanitary  inspection  of  the  sources  of  supply  is  more  important,  in  determin- 
ing the  safety  of  the  supply  from  a  sanitary  point  of  view,  than  a  chemical 
or  bacteriological  examination.  The  writer  has  for  some  years  past  enter- 
tained the  same  opinion.  Kruse  says,  however,  that  for  the  control  of  fil- 
tering plants  bacteriological  "counting-methods"  are  indispensable.  He 
also  ascribes  a  "high  scientific  value"  to  investigations  relating  to  the  pres- 
ence of  the  more  important  pathogenic  bacteria;  but  says  that,  notwith- 
standing the  improvements  in  methods  of  research,  we  cannot  wait  for  a 
demonstration  of  the  presence  of  the  cholera  or  typhoid  bacteria  before  con- 
demning a  water  as  probably  unsafe,  if  sources  of  contamination  are  dis- 
covered— or,  we  would  add,  if  cases  of  cholera  or  typhoid  fever  can  be  traced 
with  a  fair  degree  of  certainty  to  the  use  of  water  from  a  given  source. 

Fischer  (1894),  in  his  account  of  the  researches  made  during  the  Plankton 
expedition,  has  given  a  summary  of  the  experimental  evidence  relating  to  the 
presence  of  bacteria  in  the  waters  of  the  ocean.  The  species  found  were  for 
the  most  part  different  from  those  found  in  lakes  and  rivers,  and  at  some 
distance  from  the  shore  none  of  the  previously  known  species  of  micrococci 
and  bacilli  were  encountered.  The  number  of  bacteria  in  samples  from  the 
surface  at  a  distance  from  the  shore  was  comparatively  small  (usually  less 
than  five  hundred  per  cubic  centimetre),  but  in  the  vicinity  of  land  very 
large  numbers  were  sometimes  found.  At  a  distance  of  ten  metres  below  the 
surface  the  number  found  was  greatly  in  excess  of  the  number  at  the  surface 
— the  difference  being  probably  due  to  the  germicidal  action  of  sunlight.  At 
depths  of  four  hundred  metres  bacteria  were  constantly  found  in  great  num- 
bers, and  water  from  a  depth  of  eleven  hundred  metres  was  still  found  to 
contain  them. 

41 


III. 

BACTERIA    IN  THE  SOIL. 

SURFACE  soil,  and  especially  that  which  is  rich  in  organic  matter, 
contains  very  numerous  bacteria  of  many  different  species.  Some  of 
these  are  of  special  interest  on  account  of  their  pathogenic  power. 
Thus  the  bacillus  of  malignant  oedema  and  the  bacillus  of  tetanus 
have  been  shown  to  be  widely  distributed  species,  which  have  been 
obtained  by  investigators  in  various  parts  of  the  world  by  inoculating 
susceptible  animals — guinea-pigs  or  mice — with  a  little  rich  surface 
soil.  Other  species  are  interesting  because  of  their  action  in  nitrifi- 
cation and  in  the  destructive  decomposition  of  organic  material  by 
which  it  is  fitted  for  assimilation  by  the  higher  plants.  Many  of  the 
bacteria  present  in  the  soil  are  strictly  anaerobic,  and  in  attempts  to 
estimate  the  number  and  kind  of  microorganisms  present  in  a  given 
sample  this  fact  must  be  kept  in  view. 

The  simplest  method  of  studying  the  bacteria  in  the  soil  consists 
in  introducing  a  small  quantity  into  liquefied  gelatin  in  test  tubes, 
and,  after  carefully  crushing  it  with  a  sterilized  glass  rod  and  thor- 
oughly mixing  it  with  the  gelatin,  making  roll  tubes  in  the  usual 
way.  Some  of  these  should  be  put  up  for  anaerobic  cultures — i.e., 
the  tube  should  be  filled  with  an  atmosphere  of  hydrogen  according 
to  Frankel's  method.  If  the  object  in  view  is  to  estimate  the  num- 
ber of  bacteria  in  a  given  sample  of  soil  the  difficulty  is  encountered 
that,  however  finely  crushed,  the  little  masses  of  earth  are  likely  to 
contain  numerous  bacteria,  and  we  cannot  safely  assume  that  each 
colony  originates  from  a  single  germ.  Thoroughly  washing  a  small 
quantity  of  soil,  by  agitation,  in  a  considerable  quantity  of  distilled 
water,  and  then  adding  a  definite  quantity  of  the  water  to  nutrient 
gelatin  and  making  roll  tubes  or  plates,  as  in  water  analysis,  sug- 
gests itself  as  a  simple  method  ;  but  Frankel  has  shown  that  it  is  far 
from  being  reliable  when  the  object  is  to  estimate  the  number  of 
bacteria.  He  obtained  more  uniform  and  accurate  results  by  intro- 
ducing the  earth  at  once  into  liquefied  gelatin  and  crushing  it  as 
thoroughly  as  possible  with  a  strong  platinum  wire,  after  which  as 
thorough  a  mixture  as  possible  was  effected  by  tilting  the  tube  up 


BACTERIA   IN   THE   SOIL.  643 

and  down.  But  for  the  purpose  of  obtaining  pure  cultures  from  sin- 
gle colonies  of  the  various  species  present,  we  should  prefer  to  wash 
the  earth  in  distilled  water  and  to  allow  the  sediment  to  settle  before 
taking  a  portion  of  the  water  to  add  to  the  nutrient  medium. 

In  some  experiments  made  in  1881  Koch  ascertained  that  in  soil 
which  had  not  been  disturbed  but  few  bacteria  were  to  be  found  at 
the  depth  of  a  metre;  and  this  fact  has  since  been  established  by  the 
extended  researches  of  Frankel,  who  devised  a  special  boring  instru- 
ment for  obtaining  samples  of  earth  from  different  depths.  Miquel, 
in  1879,  estimated  the  number  of  bacteria  in  one  gramme  of  earth 
collected  in  the  park  of  Montsouri,  Paris,  at  a  depth  of  twenty  centi- 
metres, at  700,000;  and  in  a  cultivated  field  which  had  been  treated 
with  manure,  at  900,000.  The  following  results  were  obtained  by 
Adametz  :  One  gramme  of  earth  from  a  sandy  soil  contained  at  the 
surface  380,000,  at  a  depth  of  twenty  to  twenty-five  centimetres 
400,000 ;  the  same  quantity  of  clayey  soil  contained  at  the  surface 
500,000,  at  a  depth  of  twenty  to  twenty-five  centimetres  460,000. 

In  experiments  made  by  Beumer  (1886)  and  by  Maggiora  (1887) 
considerably  greater  numbers  were  found,  but  the  last-named  ob- 
server, in  some  instances  at  least,  kept  the  earth  for  some  time  after 
collecting  it,  which  may  have  materially  influenced  the  result. 
Beumer  obtained  from  a  specimen  of  sandy  humus  taken  from  a 
depth  of  three  metres  45,000,000  to  the  gramme ;  at  four  metres, 
10,000,000;  at  five  metres,  8,000,000;  at  six  metres,  5,000,000. 
These  specimens  were  obtained  from  the  vicinity  of  hospitals  at 
Greifswald.  In  a  churchyard,  at  a  depth  of  four  metres,  the  num- 
ber in  one  experiment  was  1,152,000,  and  in  another  1,278,000. 

Frankel  has  given  special  attention  to  the  examination  of  undis- 
turbed soil  not  in  the  immediate  vicinity  of  dwellings.  In  samples 
from  a  fruit  orchard  near  Potsdam  he  found  that  the  superficial 
layers  contained  from  50,000  to  350,000  germs  per  cubic  centimetre. 
The  greatest  number  was  not  immediately  upon  the  surface,  but  at 
from  one-quarter  to  one-half  metre  below  the  surface.  The  num- 
ber was  found  to  be  greater  in  summer  than  in  winter,  the  maximum 
being  in  July  and  August.  At  a  depth  of  three-quarters  of  a  metre 
to  a  metre  and  a  half  there  was  a  very  great  and  abrupt  diminution  in 
the  number  of  germs.  From  200, 000  at  one-half  metre  the  number  fell 
to  2,000  at  a  depth  of  a  metre,  from  250,000  at  three-quarters  of  a 
metre  to  200  at  one  metre,  etc. ,  and  at  a  depth  of  one  and  one-half 
metres,  in  some  instances,  no  more  living  germs  were  obtained.  In 
other  experiments  a  few  colonies  developed  from  earth  obtained  at  a 
depth  of  three  or  four  metres,  but  these  were  slow  in  making  their 
appearanc'e,  and  often  several  days,  or  even  weeks,  elapsed  before 
they  became  visible  in  Esmarch  roll  tubes.  In  experiments  with  sur- 


G44  BACTERIA   IN   THE   SOIL. 

face  soil,  on  the  contrary,  a  multitude  of  colonies  developed  within 
twenty-four  to  forty-eight  hours,  and,  as  many  liquefying  bacteria 
were  present,  it  was  necessary  to  make  the  enumeration  on  the  first 
or  second  day,  at  which  time,  no  doubt,  many  of  the  bacteria  present 
had  not  yet  formed  visible  colonies.  The  results  obtained  have, 
therefore,  only  a  relative  value. 

The  most  important  fact  developed  by  FrankePs  researches  is  that 
in  virgin  soil  there  is  a  dividing  line  at  a  depth  of  from  three-quarters 
to  one  and  one-half  metres,  below  which  very  few  bacteria  are  found, 
and  that,  consequently,  the  "  ground- water  region  "  is  free  from  micro- 
organisms, or  nearly  so,  notwithstanding  the  immense  numbers  pre- 
sent in  the  superficial  layers. 

The  extended  researches  of  Maggiora,  made  in  the  vicinity  of 
Turin,  led  him  to  the  following  conclusions  : 

1.  The  number  of  germs  in  desert  and  forest  soils  is  much  smaller,  other 
conditions  being-  equal,  than  in  cultivated  lands,  and  in  these  it  is  less  than 
in  inhabited  localities. 

2.  In  desert  soils  the  number  of  germs  bears  a  relation  (a)  to  the  geologi- 
cal epoch  to  which  the  lands  belong,  and,  within  certain  limits,  to  the  height 
above  the  level  of  the  sea — the  older  the  soil  and  the  greater  the  altitude, 
other  things  being  equal,  the  fewer  the  germs  ;  (&)  to  the  compactness  and 
aeration  of  the  soil — the  more  compact  and  impermeable  to  air  the  smaller 
the  number  of  germs  capable  of  developing  in  gelatin  ;  (c)  to  the  nature  of 
the  soil — sandy  soils  contain  fewer  germs  than  soils  rich  in  clay  and  in 
humus. 

3.  In  cultivated  lands  the  number  of  germs  augments  with  the  activity 
of  cultivation  and  the  strength  of  the  fertilizers  used. 

4.  In  inhabited  localities  the  number  of  germs  in  the  superficial  layers  is 
very  great.     In  the  deep  layers  it  usually  diminishes  rapidly,  as  is  the  case 
in  all  other  soils. 

As  to  the  kinds  of  bacteria  present,  and  their  biological  characters 
and  functions  in  preparing  organic  material  for  assimilation  by  the 
plants  whose  roots  penetrate  the  soil,  we  have  yet  much  to  learn. 
Frankel  remarks  that  the  species  most  frequently  encountered  in  the 
deeper  strata  of  the  soil  were  three  bacilli  which  also  abound  in  the 
superficial  layers — viz.,  the  "  hay  bacillus,"  the  "wurzel  bacillus," 
and  the  "hirnbacillus."  In  all  eleven  bacilli  were  isolated  and  cul- 
tivated. Micrococci  were  only  found  four  times,  and  spirilla  not  at 
all.  Mould  fungi  were  more  abundant,  and  especially  one  previously 
obtained  from  the  air  by  Hesse  and  called  by  him  "brauner  Schim- 
melpilz."  Anaerobic  bacilli,  contrary  to  expectation,  were  not  ob- 
tained in  Frankel's  researches,  and  no  pathogenic  species  were  found 
in  the  deeper  layers  of  the  soil.  We  have  already  referred  to  the 
fact  that  the  bacillus  of  malignant  oedema  and  the  bacillus  of  tetanus, 
two  pathogenic,  anaerobic  species,  are  common  in  rich  surface  soil  in 
various  parts  of  the  world. 


BACTERIA   IX   THE    SOIL.  645 

The  results  obtained  in  the  researches  referred  to,  in  which  nutri- 
ent gelatin  was  used  as  a  culture  medium,  are  no  doubt  very  in- 
complete, not  only  on  account  of  the  liquefaction  of  the  gelatin  by 
common  liquefying  bacilli  before  other  species  present  have  formed 
visible  colonies,  but  also  because  this  is  not  a  favorable  culture  me- 
dium for  some  of  the  species  present  in  the  soil.  Thus  Frankland  has 
succeeded  in  isolating  a  nitrifying  ferment  which  he  calls  "  Bacillo- 
coccus,"  which  grows  abundantly  in  bouillon,  but  fails  to  grow  in 
nutrient  gelatin.  Winogradski  has  also  obtained  in  pure  cultures  a 
nitrifying  ferment  from  the  soil  in  the  vicinity  of  Zurich,  which  he 
has  called  "  Nitromonas." 

Comparatively  few  micrococci  are  found  in  the  soil,  while  in  the 
air  they  are  usually  found  to  be  more  abundant  than  bacilli.  This 
is  perhaps  due  to  the  fact  that  the  bacilli  are  more  promptly  destroyed 
by  desiccation  and  the  action  of  sunlight. 

Several  bacteriologists  have  made  investigations  relating  to  the 
duration  of  vitality  of  pathogenic  bacteria  in  the  soil.  Frankel  found 
that  in  Berlin  the  bacillus  of  anthrax,  in  Esmarch  roll  tubes,  when 
buried  in  the  soil  at  a  depth  of  two  metres,  only  occasionally  gave 
evidence  of  growth,  and  at  three  metres  no  development  occurred. 
The  comparatively  low  temperature  at  this  depth  was  no  doubt  an 
important  factor  in  influencing  the  result.  The  cholera  spirillum  in 
the  months  of  August,  September,  and  October  grew  at  a  depth  of 
three  metres,  but  in  the  remaining  months  of  the  year  failed  to  grow 
at  two,  while  growth  occurred  at  one  and  one-half  metres.  The 
bacillus  of  typhoid  fever  grew  at  three  metres  during  the  greater 
portion  of  the  year. 

Giaxa  has  made  extended  and  interesting  experiments  with  the 
cholera  spirillum,  cultures  of  which  he  added  to  different  kinds  of 
soil  (garden  earth,  clay,  sand)  and  placed  at  different  depths  below 
the  surface — one-quarter,  one -half,  and  one  metre.  Some  of  the  earth 
was  sterilized  and  some  was  not.  In  the  unsterilized  earth  he  found 
the  cholera  spirillum  in  considerable  numbers  at  the  end  of  twenty- 
four  hours  at  the  greatest  depth  tested  (one  metre),  but  at  the  end  of 
forty  eight  hours  it  had  disappeared  in  five  experiments  out  of  seven 
— the  lowest  temperature  at  this  depth  was  20°  C.  In  the  sterilized 
soil  the  result  was  different ;  the  cholera  spirillum  was  present  in 
enormous  numbers  at  the  end  of  four  days  at  a  depth  of  a  metre, 
and  was  still  found  in  smaller  numbers  at  the  end  of  twelve  days,  but 
had  disappeared  at  the  end  of  twenty-one  days.  These  results  indicate 
that  the  presence  of  common  saprophytes  in  the  soil  is  prejudicial  to 
the  development  of  the  cholera  spirillum,  and  that  under  ordinary 
circumstances  it  succumbs  in  the  struggle  for  existence  with  these 
more  hardy  microorganisms. 


C46  BACTERIA   IN    THE   SOIL. 

The  researches  of  Proskauer  (1891)  confirm  those  of  Frankel  and 
others  as  to  the  rapid  diminution  in  the  number  of  bacteria  in  the 
deeper  layers  of  the  soil.  They  also  agree  with  those  of  Gartner  in 
showing  that  in  the  soil  of  churchyards  the  number  of  bacteria 
diminishes  greatly  in  the  soil  beneath  the  layer  containing  coffins. 
In  general  the  influence  of  dead  bodies  upon  the  bacteria  in  the  soil 
in  the  vicinity  of  coffins  was  very  slight;  in  the  subsoil  of  the  grave- 
yard there  were  not  many  more  bacteria  than  in  similar  soil  outside 
of  this.  Reimers  had  previously  shown  that  samples  of  earth  from 
two  graves,  in  one  of  which  the  body  had  been  buried  for  thirty-five 
years  and  in  the  other  for  one  and  one-half  years,  gave  similar  re- 
sults when  examined  by  bacteriological  methods. 

Manfredi  in  1892  published  the  results  of  his  extended  investiga- 
tions relating  to  the  dust  in  the  streets  of  Naples,  The  number  of 
bacteria  varied  greatly  in  different  parts  of  the  city.  In  streets 
where  the  traffic  was  least  and  hygienic  conditions  the  best  the 
average  number  was  10,000,000  per  gramme.  In  dirty  and  busy 
thoroughfares  the  average  was  1,000,000,000,  and  in  certain  locali- 
ties the  number  was  even  five  times  as  great  as  this.  Injections  into 
guinea-pigs  gave  a  positive  result  in  seventy-three  per  cent  of  the 
animals  experimented  upon.  Among  the  known  pathogenic  bacte- 
ria obtained  in  this  way  were  the  pus  cocci  (in  eight),  Bacillus  tuber- 
culosis (in  three),  the  bacillus  of  malignant  oedema,  and  the  tetanus 
bacillus. 

In  the  memoir  of  Fulles  (1891)  the  following  species  are  described 
as  having  been  found  by  him  in  the  soil  at  Freiburg,  Germany: 


MICROCOCCI. 

(a)  Non-liquefying. — Micrococcus  aurantiacus  (Colin),  Micrococcus  can- 
didus  (Cohn),  Micrococcus  luteus  (Cohn),  Mjcrococcus  candicans  (Fliigge), 
Micrococcus  versicolor  (Fliigge),  Micrococcus  cinnabareus  (Fliigge),  Micro- 
coccus  cereus  albus  (Passet),  Micrococcus  fervitosus  (Adametz),  Bother  coc- 
cus (Maschek*. 

(b)  Liquefying. — Micrococcus  flavus  liquefaciens  (Fliigge),  Micrococcus 
flavus  desidens  (Fliigge),  Diplococcus  luteus  (Adametz),  Sarcina  lutea. 

NON-PATHOGENIC   BACILLI. 

(a)  Non-liquefying. — Bacillus  fluorescens  putidus  (Fliigge),  Bacillus  mus- 
coides  (Liborius),  Bacillus  scissus  (Frankland),  Bacillus  candicans,  Bacillus 
diffusus  (Frankland),  Bacillus  filiformis  (Tils),  Bacillus  luteus  (Fliigge), 
Fluorescent  water  bacillus  (Eisenberg),  Bacillus  viridis  pallescens  (Frick), 
Bluish-green  fluorescent  bacillus  (Adametz),  Bacillus  stolonatus  (Adametz), 
Bacillus  Ziirnianum  (List),  Bacillus  aerogenes  (Miller),  Bacillus  No.  1  and 
Bacillus  No.  2  (Fulles). 

(6)  Liquefying.—  Bacillus  ramosus  liquefaciens  (Flugge\  Bacillus  liqui- 
dus  (Frankland),  Bacillus  ramosus— "wurzel  bacillus,"  Bacillus  subtilis 


BACTERIA  IN  THE   SOIL.  G47 

(Ehrenberg),  Bacillus  mesentericus  fuscus  (Fliigge),  Bacillus  mesentericus 
vulgatus  (Fliigge),  Bacillus  fluorescens  liquefaciens  (Fliigge),  Lemon-yellow 
bacillus  (Maschek),  Green  yellow  bacillus  (Eisenberg),  Gas-forming  bacillus 
(Eisenberg),  Gray  bacillus  (Maschek),  Bacillus  prodigiosus  (Ehrenberg), 
Proteus  mirabilis  (Hauser),  Proteus  vulgaris  (Hauser),  Bacillus  mesentericus 
vulgatus,  Bacillus  cuticularis  (Tils),  "  Weisser  bacillus"  (Eisenberg). 

(c)  Pathogenic. — Bacillus  oedematis  maligni  (Koch). 

In  addition  to  the  above  the  following  species  have  been  described  by 
other  authors:  Bacillus  liquefaciens  magnus  (Liideritz),  Bacillus  radiatus 
(Liideritz),  Bacillus  solidus  (Liideritz),  Bacillus  mycoides  roseus  (Scholl), 
Bacillus  viscpsus  (Frankland),  Bacillus  candicans  (Frankland),  Bacillus 
poliformis  (Liborius),  Clostridium  fcetidum  (Liborius). 

Pathogenic  species. — Staphylococcus  pyogenes  aureus  (Rosenbach),  Ba- 
cillus tetani(Nicolaier),  Streptococcus  septicus  (Nicolaier),  Pseudo-cedema  ba- 
cillus (Liborius),  Bacillus  septicus  agrigenus  (Nicolaier),  Bacillus  of  Utpadel. 


IV. 

BACTERIA  OF  THE  SURFACE  OF  THE  BODY  AND  OF 
EXPOSED  MUCOUS  MEMBRANES. 

GREAT  numbers  of  bacteria  of  various  species  multiply  upon  the 
surface  of  the  human  body,  where  they  find  the  necessary  pabulum 
in  the  excretions  from  the  skin  and  the  exfoliated  epithelium.  Evi- 
dently the  number  will  be  largely  influenced  by  the  clothing  worn, 
the  atmospheric  conditions  as  to  heat  and  moisture,  personal  habits, 
etc.  The  writer  has  frequently  inoculated  culture  media  with  a  drop 
of  sterilized  fluid  which  had  been  placed  upon  the  surface  of  the  body 
of  patients  in  hospitals  and  of  healthy  persons.  By  friction  with  a 
platinum  needle  at  the  point  where  the  drop  of  fluid  is  applied  the 
surface  is  washed  and  a  little  epithelium  detached.  Cultures  may 
always  be  obtained  by  inoculating  nutrient  media  from  a  drop  of  fluid 
applied  in  this  way.  Micrococci  of  various  species,  including  the  pus 
cocci,  are  very  commonly  encountered  ;  sarcinse  and  various  bacilli 
are  also  frequently  met  with.  Even  the  hands,  which  by  reason  of 
their  exposure  and  frequent  ablutions  are  freer  from  exfoliated  epi- 
thelium than  portions  of  the  body  covered  with  clothing,  have  con- 
stantly attached  to  their  surface  a  considerable  number  of  bacteria. 
This  is  shown  by  the  experiments  of  Kiimmel  and  Forster,  of  Fiir- 
bringer  and  others,  with  reference  to  the  disinfection  of  the  hands. 
Forster  found  that  after  the  most  careful  cleaning  of  the  hands  with 
soap,  water,  and  a  brush,  contact  of  the  fingers  with  nutrient  gelatin 
always  resulted  in  the  development  of  a  greater  or  less  number  of 
colonies. 

Bordoni-Uffreduzzi,  in  his  researches  relating  to  the  bacteria  of 
the  skin,  obtained  in  pure  cultures  five  different  species  of  micrococci 
and  two  bacilli.  Pure  cultures  of  his  Bacterium  graveolens,  which 
was  usually  found  between  the  toes,  gave  off  a  disagreeable  odor  like 
that  observed  from  this  locality  in  certain  individuals.  In  his  re- 
searches made  in  Havana  the  writer  frequently  encountered  in  cul- 
tures from  the  surface,  associated  with  various  micrococci,  his  Micro- 
coccus  tetragenus  versatilis. 

Fiirbringer  found  quite  frequently  in  the  spaces  beneath  the  fin- 


BACTERIA  OF  THE  SURFACE  OF  THE  BODY.        649 

ger  nails  Staphylococcus  pyogenes  aureus  associated  with  various 
other  microorganisms.  A  similar  result  had  previously  been  reported 
by  Bockhart. 

In  his  examinations  of  water  from  various  sources  Miquel  found 
that  "wash-water"  from  the  floating  laundries  on  the  Seine  con- 
tained more  bacteria  than  water  from  any  other  source,  even  than 
the  water  of  the  Paris  sewers.  His  enumeration  gave  twenty-six 
million  germs  per  cubic  centimetre. 

Hohein  has  enumerated  the  colonies  developing  from  undercloth- 
ing worn  for  various  lengths  of  time  and  made  of  different  kinds  of 
material.  A  piece  of  the  goods  to  be  tested  was  sewed  fast  to  the 
underclothing,  so  as  to  come  in  immediate  contact  with  the  body  ;  at 
the  end  of  a  given  time  a  fragment  one-quarter  of  a  centimetre  square 
was  cut  up  as  fine  as  possible  and  distributed  in  nutrient  gelatin. 
Plates  were  made  and  the  colonies  counted  at  the  end  of  five  or  six 
days. 

In  an  experiment  in  which  sterilized  woven  goods  were  worn  next 
to  the  skin  of  the  upper  arm  the  following  results  were  obtained  : 
Linen  goods,  at  the  end  of  one  day  28,  two  days  4,180  colonies  ;  cot- 
ton goods,  end  of  one  day  105,  end  of  two  days  1,870  ;  woollen  goods, 
end  of  one  day  606,  end  of  two  days  6,799.  When  the  material  had 
been  in  contact  with  the  skin  for  four  days  the  colonies  which  devel- 
oped were  so  numerous  that  they  could  not  be  counted. 

Maggiora  isolated  twenty-two  species  of  bacteria  from  his  cultures 
inoculated  with  epidermis  from  the  foot.  None  of  these  proved  to 
be  pathogenic  for  mice,  rabbits,  or  guinea-pigs.  Several  gave  off  a 
strong  odor  of  trimethylamin,  similar  to  that  of  sweating  feet. 

The  following  species  have  been  found  upon  the  surface  of  the 
body : 

Non-pathogenic. — Diplococcus  albicans  tardus  (Unna  and  Tommasoli), 
Diplococcus  citreus  liquefacieiis  (Unna  and  Tommasoli),  Diplococcus  flavus 
liquefaciens  tardus  (Unna  and  Tommasoli),  Staphylococcus  viridis  flaves- 
cens  (Guttmann),  Bacillus  graveolens  (Bordoni-Uffreduzzi),  Bacillus  epider- 
midis  (Bordoni),  Ascobacillus  citreus  (Unna  and  Tommasoli),  Bacillus  fluo- 
rescens  liquefaciens  minutissimus  (Unna  and  Tommasoli),  Bacillus  aureus 
(Unna  and  Tommasoli),  Bacillus  ovatus  minutissimus  (Unna  and  Tomma- 
soli), Bacillus  albicans  pateriformis  (Unna  and  Tommasoli),  Bacillus  spini- 
ferus  (Unna  and  Tommasoli),  Bacillus  of  Scheurlen,  Micrococcus  tetragenus 
versatilis  (Sternberg),  Bacillus  Havaniensis  liquefaciens  (Sternberg). 

Pathogenic.  —Staphylococcus  pyogenes  albus,  Staphylococcus  pyogenes 
aureus,  Streptococcus  pyogenes,  Diplococcus  of  Demme,  Bacillus  of  L  emme, 
Bacillus  of  Schimmelbusch,  Bacillus  of  Tommasoli,  Bacillus  saprogenes  II. 
(Rosenbach) ,  Bacillus  parvus  ovatus  (Loftier). 

SURFACE   OF   MUCOUS   MEMBRANES. 

Cultures  made  from  the  conjunctivas  of  healthy  persons  usually 
show  the  presence  of  various  micrococci,  and  sometimes  of  bacilli. 


650         BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

McFarland  (1895)  says  that  in  his  researches  the  microorganisms 
found  were  for  the  most  part  "those  already  described  by  others  and 
of  common  occurrence  in  the  air."  He  encountered,  however,  sev- 
eral bacilli  not  previously  described  ("  Bacillus  hirsutus,  Bacillus 
ccerulefaciens,  Bacillus  circurnscriptus,  Bacillus  succinacius,  Bacillus 
violaceus  flavus").  Lachowicz  (1895)  failed  to  obtain  any  bacteria 
in  his  cultures  from  the  con junctival  sac  in  sixty-nine  per  cent  of  the 
healthy  eyes  examined  by  him  (sixty-three  eyes  in  all).  He  con- 
cludes that  the  microorganisms,  which  at  times  are  found  in  the 
healthy  conjunctival  sac,  come  principally  from  the  air;  that  they 
are  present  in  small  numbers  and  probably  remain  only  for  a  short 
time.  His  experiments  show  that  most  species  when  artificially 
introduced  rapidly  diminish  in  numbers  and  soon  disappear  entirely. 
Cultures  of  Streptococcus  pyogenes  and  of  Bacillus  xerosis  conjunc- 
tive introduced  into  healthy  eyes  did  not  cause  the  slightest  irrita- 
tion. In  this  connection  we  may  remark  that  the  same  is  true  as 
regards  pathogenic  bacteria  introduced  into  the  bladder,  but  that 
when  there  is  some  cause  of  local  irritation  or  injury  a  chronic 
cystitis  is  likely  to  be  developed.  In  like  manner,  we  believe,  chronic 
conjunctivitis  may  be  developed  as  the  result  of  local  irritation  in 
connection  with  the  presence  of  pathogenic  bacteria  and  especially  of 
the  pyogenic  micrococci. 

The  extended  researches  of  Bach  (1894)  gave  results  corresponding 
with  those  of  previous  investigators,  and  not  with  those  reported  by 
Lachowicz,  who,  as  stated  above,  failed  to  obtain  cultures  from  sixty- 
nine  per  cent  of  the  healthy  eyes  examined.  Bach  says :  "  In  a  large 
percentage  of  the  cases  the  presence  of  bacteria  may  be  demonstrated, 
even  when  the  conjunctiva  presents  a  perfectly  normal  appearance; 
the  conjunctival  sac  must  therefore  be  regarded  as  constantly  in- 
fected." Bach  describes  twenty-seven  different  microorganisms  ob- 
tained by  him  in  pure  cultures  from  this  source,  of  these  eighteen 
are  micrococci.  He  recognizes  the  fact  that  most  of  them  come  from 
the  air,  while  others  are  introduced  by  the  hands  in  rubbing  the 
eyes,  etc.  In  diseased  conditions  these  are  more  numerous  than  in 
health,  but  the  pus  cocci  are  not  infrequently  found  in  healthy  eyes. 

As  bacteria  are  constantly  present  in  the  air,  they  are  necessarily 
deposited  upon  the  moist  mucous  membrane  of  the  nose  during  in- 
spiration. Indeed,  it  would  appear  as  if  an  important  function  of 
this  extended  mucous  membrane  is  to  purify  the  air  from  suspended 
particles,  and  it  has  been  shown  by  experiment  that  expired  air  is 
practically  free  from  bacteria.  The  greater  number  of  those  con- 
tained in  inspired  air  are  deposited  upon  the  mucous  membrane  of 
the  anterior  nares.  In  culture  experiments  made  by  Von  Besser, 
Wright,  and  others  the  nasal  mucus  was  found  to  contain  a  great 


AND  OF  EXPOSED  MUCOUS  MEMBHANES.  651 

variety  of  bacteria;  among  others  the  pus  cocci  were  frequently 
found  by  both  of  the  observers  mentioned.  In  eighty  one  cases  Von 
Besser  found  the  "  diplococcus  pneumonias  "  fourteen  times,  Staphy- 
lococcus  pyogenes  aureus  fourteen  times,  Streptococcus  pyogenes 
seven  times,  and  Friedlander's  bacillus  twice.  Twenty-eight  of  the 
cases  examined  were  convalescents  in  hospital;  among  these  the 
pathogenic  species  mentioned  were  found  less  frequently  than  in  other 
individuals.  The  following  non-pathogenic  species  were  isolated: 
Micrococcus  liquefaciens  albus  in  twent}*-two  cases,  Micrococcus  al- 
bus  in  nine  cases,  Micrococcus  cumulatus  tenuis  in  fourteen  cases, 
Micrococcus  flavus  liquefaciens  in  three  cases,  Bacillus  striatus  albus 
in  ten  cases,  etc. 

Paulsen  (1890)  made  thirty-one  cultures  in  nutrient  gelatin  from 
sixteen  persons  and  thirty-three  in  nutrient  agar  from  twenty-two 
persons,  with  the  following  result :  Eleven  remained  sterile,  nineteen 
showed  not  more  than  ten  colonies,  sixteen  less  than  one  hundred, 
twelve  more  than  one  hundred,  and  in  six  the  number  was  so  great 
that  they  could  not  be  counted.  Micrococci  were  more  numerous 
than  bacilli ;  of  these  a  "  sulphur-yellow  coccus  "  in  tetrads  was  found 
in  eight  individuals.  Various  species  of  liquefying  cocci,  resem- 
bling the  pus  cocci,  were  isolated,  but  the  conclusion  was  reached 
that  none  of  these  were  identical  with  the  staphylococci  of  pus, 
which  Von  Besser  and  Wright  both  found  in  a  considerable  propor- 
tion of  the  culture  experiments  made  by  them. 

Thomson  and  Hewlett  (1895)  have  recently  reported  results  which 
differ  to  some  extent  from  those  previously  reported.  While  they 
found  numerous  bacteria  in  the  vestibulum  naris,  cultures  made  from 
mucus  obtained  from  the  interior  of  the  nose  usually  gave  a  negative 
result — sixty-four  out  of  seventy-six  remained  absolutely  sterile, 
while  in  seven  there  was  a  scanty  growth  only.  They  conclude  that 
while  microorganisms  are  occasionally  found  upon  the  Schneider- 
ian  membrane  they  are  not  numerous  and  are  often  entirely  absent; 
and  that  the}*  are  rarely  found  upon  the  pituitary  membrane.  Straus 
(1895)  has  examined  the  nasal  secretions  of  persons  associated  with 
tubercular  patients  for  the  purpose  of  ascertaining  if  the  tubercle  ba- 
cillus was  present.  The  presence  of  this  bacillus  was  demonstrated, 
by  inoculation  into  guinea-pigs,  in  nine  healthy  individuals  out  of 
twenty -nine  examined ;  two  of  these  were  physicians  and  six  were 
nurses. 

Very  extended  researches  have  been  made  with  reference  to  the 
bacteria  present  in  the  human  mouth,  which  show  that  numerous 
species  are  constantly  present  in  the  buccal  secretions  and  upon  the 
surface  of  the  moist  mucous  membrane.  Some  of  these  are  occa- 
sional and  accidental,  while  others  appear  to  have  their  normal  habi- 


652 

tat  in  the  mouth,  where  the  conditions  as  to  temperature,  moisture, 
and  presence  of  organic  pabulum  are  extremely  favorable  for  their 
development.  A  minute  drop  of  saliva  spread  upon  a  glass  slide, 
dried,  and  stained  with  one  of  the  aniline  colors,  will  always  be 
found  to  contain  an  immense  number  of  bacteria  of  various  forms. 
Some  of  these  are  attached  to  epithelial  cells  and  some  scattered  about 
singly  or  in  groups.  Among  those  seen  in  a  single  specimen  we  will 
usually  find  cocci  in  tetrads,  in  chains,  and  in  irregular  groups, 
bacilli  of  various  dimensions,  and  occasionally  spirilla.  According 
to  Prof.  Miller,  of  Berlin,  the  following  species  almost  invariably 
occur  in  every  mouth :  Leptothrix  innominata,  Bacillus  buccalis  max- 
imus,  Leptothrix  buccalis  maxima,  lodococcus  vaginatus,  Spirillum 
sputigenum,  Spirochaate  dentium.  All  of  these  fail  to  grow  in  ordi- 
nary culture  media.  Miller  has  made  extended  attempts  to  obtain 
cultures  by  varying  the  medium  used  and  attempting  to  imitate  as 
nearly  as  possible  the  natural  medium  in  which  they  are  found;  but 
his  attempts  have  been  unsuccessful,  or  nearly  so — "  only  line  cultures 
afforded  a  limited  growth,  but  the  colonies  never  developed  more 
than  fifteen  to  twenty  cells,  and  a  transference  to  a  second  plate 
proved  futile,  no  further  growth  taking  place." 

Up  to  the  year  1885  Miller  had  isolated  twenty-two  different 
species  of  bacteria  from  the  human  mouth.  Ten  of  these  were  cocci, 
five  short  bacilli,  six  long  bacilli,  and  one  a  spirillum.  Later  the 
same  author  cultivated  eight  additional  species.  Vignal  has  isolated 
and  described  seventeen  species  obtained  by  him  in  pure  cultures 
from  the  healthy  human  mouth;  most  of  these  are  bacilli,  and  Miller, 
who  found  micrococci  to  be  more  numerous,  supposes  the  difference 
in  results  to  be  due  to  the  fact  that  many  of  the  cocci  do  not  grow  in 
nutrient  gelatin,  which  was  the  medium  employed  by  Vignal.  In 
the  researches  of  the  last-named  author  the  following  species  were 
obtained  most  frequently,  in  the  order  given:  1.  Bacterium  termo. 
2.  Bacillus  e  (Bacillus  ulna  ?).  3.  Potato  bacillus.  4.  Coccus  a.  5. 
Bacillus  b.  6.  Bacillus  d.  7.  Bacillus  c  (Bacillus  alvei  ?).  8.  Bacil- 
lus subtilis.  9.  Staphylococcus  pyogenes  albus.  10.  Staphylococcus 
pyogenes  aureus. 

Among  the  species  above  enumerated  we  find  two  of  the  most 
common  pus  cocci,  Staphylococcus  albus  and  aureus.  but  no  mention 
is  made  of  another  important  pathogenic  micrococcus  which  is  fre- 
quently found  in  the  healthy  human  mouth,  viz.,  the  micrococcus  of 
sputum  septicaemia,  first  named  by  the  writer  Micrococcus  Pasteuri. 
This  does  not  grow  at  ordinary  temperatures,  and  consequently 
would  not  be  obtained  in  gelatin  plate  cultures.  Very  different  re- 
sults have  been  reported  by  different  observers  as  to  the  frequency 
with  which  the  pathogenic  cocci  are  found  in  the  buccal  cavity. 


AND  OF  EXPOSED  MUCOUS  MEMBRANES.  053 

Black  found  in  the  saliva  of  ten  healthy  individuals  the  Staphy- 
lococcus  pyogenes  aureus  seven  times,  Staphylococcus  pyogenes  al- 
bus  four  times,  and  Streptococcus  pyogenes  three  times.  On  the 
other  hand,  Netter  found  Staphylococcus  aureus  only  seven  times  in 
one  hundred  and  twenty-seven  individuals  examined.  Miller  also 
has  rarely  found  the  pus  cocci  in  the  mouths  of  healthy  persons. 
Streptococcus  pyogenes  was  not  found  by  Vignal  in  his  extended 
researches.  The  experiments  of  the  writer,  of  Vulpian,  Frankel, 
letter,  Claxton,  and  others  show  that  the  micrococcus  which  in 
1885  I  named  Micrococcus  Pasteuri,  and  which  is  identical  with  the 
"  diplococcus  pneumonia  "  of  German  authors,  is  frequently  present 
in  the  healthy  human  mouth— now  called  Micrococcus  pneumonia 
crouposaB.  Ketter  examined  the  saliva  of  one  hundred  and  sixty-five 
healthy  individuals  and  obtained  it  in  fifteen  per  cent  of  the  number 
examined. 

Another  pathogenic  micrococcus  which  is  frequently  present  in 
the  mouths  of  healthy  persons  is  the  Micrococcus  tetragenus  of  Koch. 
The  following  pathogenic  bacteria  have  also  been  isolated  and  de- 
scribed :  Bacillus  crassus  sputigenus  (Kreibohm),  Bacillus  salivarius 
septicus  (Biondi).  The  Streptococcus  septo-pyaBmicus  of  Biondi  is 
described  as  having  characters  identical  with  those  of  the  Strepto- 
coccus pyogenes  of  Rosenbach.  Two  other  pathogenic  species  de- 
scribed by  Biondi  were  each  found  in  a  single  case  only.  Miller 
has  described  the  following  pathogenic  species  isolated  and  studied 
by  him  :  Micrococcus  gingiva3  pyogenes,  Bacterium  gingivse  pyo- 
genes, Bacillus  dentalis  viridans,  Bacillus  pulpa3  pyogenes. 

Rosenthal  (1893)  examined  the  secretions  from  the  mouths  of 
fourteen  individuals  and  obtained  twenty-eight  different  bacteria;  of 
these  twenty-one  had  been  previously  described.  Five  species  be- 
lieved to  be  new  are  described  in  detail  by  Rosenthal,  viz. :  Sarcina 
viridis  flavesceris,  Micrococcus  Reessii,  Micrococcus  ochraceus,  Dip- 
lococcus Hauseri,  Bacterium  cerasinum. 

Vignal  has  tested  a  considerable  number  of  microorganisms, 
obtained  by  him  in  his  cultures  from  the  healthy  human  mouth,  with 
reference  to  their  peptonizing  action  upon  various  kinds  of  food,  with 
the  idea  that  some  of  them  may  have  an  important  physiological 
function  of  this  kind.  Out  of  nineteen  species  he  found  ten  which, 
after  a  longer  or  shorter  time,  dissolved  fibrin,  nine  which  dissolved 
gluten,  ten  which  dissolved  casein,  and  five  which  dissolved  albumin ; 
nine  changed  lactose  into  lactic  acid,  seven  inverted  cane  sugar,  seven 
caused  the  fermentation  of  glucose,  and  seven  coagulated  milk. 

Sanarelli  (1891)  has  shown  that  normal  saliva  has  the  power 
of  destroying  the  vitality  of  a  limited  number  of  certain  patho- 
genic bacteria,  including  the  following  species:  Staphylococcus 
pyogenes  aureus,  Streptococcus  pyogenes,  Micrococcus  tetragenus 


654         BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

Bacillus  typhi  abdominalis,  Spirillum  cholerse  Asiaticse.  When  to 
ten  cubic  centimetres  of  saliva,  sterilized  by  filtration  through  porce- 
lain, the  above-mentioned  pathogenic  bacteria  were  added  in  small 
numbers  by  means  of  a  platinum  needle  carried  over  from  a  pure 
culture,  no  development  occurred,  and  at  the  end  of  twenty-four 
hours  the  bacteria  introduced  were  incapable  of  growth  in  a  suitable 
medium.  But  when  this  amount  of  filtered  saliva  was  inoculated 
with  a  large  platinum  loop — an  ose — a  certain  number  of  the  bacteria 
survived,  and  at  the  end  of  three  or  four  days  an  abundant  develop- 
opment  occurred.  At  first,  however,  the  number  of  living  cells  was 
considerably  diminished.  In  saliva  to  which  one  ose  of  a  culture  of 
Staphylococcus  aureus  was  added  thirteen  thousand  eight  hundred 
and  forty  colonies  developed  in  a  plate  made  immediately  after  inocu- 
lation, while  a  plate  made  at  the  end  of  twenty-four  hours  contained 
but  one  hundred  and  thirty-two  colonies,  and  one  at  the  end  of  forty- 
eight  hours  had  but  eight  colonies.  Subsequently  multiplication 
occurred,  and  a  plate  made  on  the  ninth  day  after  inoculation  con- 
tained so  many  colonies  that  they  could  not  be  counted. 

The  diphtheria  bacillus  was  not  destroyed  in  filtered  saliva,  but 
did  not  multiply  in  it.  On  the  other  hand,  it  proved  to  be  a  very 
favorable  medium  for  the  development  of  Micrococcus  pneumonise 
crouposaB. 

Mucus  from  the  surface  of  the  meatus  urinarius  of  man  and 
woman,  or  from  the  vagina,  will  always  be  found  to  contain  various 
bacteria  ;  but  the  bladder,  the  uterus,  and  Fallopian  tubes  in  healthy 
individuals  are  free  from  microorganisms. 

Winter  has  isolated  twenty-seven  different  species  from  vaginal 
and  cervical  mucus,  and  reports  that  he  found  Staphylococcus  pyo- 
genes  albus  in  one-half  of  the  cases  examined.  A  streptococcus  was 
also  encountered  which  resembled  Streptococcus  pyogenes,  although 
not  positively  identified  with  it.  Samschin,  on  the  other  hand,  failed 
to  obtain  the  pus  cocci  in  vaginal  mucus  from  healthy  women. 

Donderlein,  Yon  Ott,  and  others  have  carefully  examined  the 
lochial  discharge  with  reference  to  the  presence  of  bacteria.  The 
first-named  author  found  that  in  healthy  women  the  lochial  discharge 
obtained  from  the  uterus  was  free  from  germs,  but  when  collected 
from  the  vagina  various  microorganisms  were  obtained.  In  one  case 
in  which  some  fever  existed  Staphylococcus  pyogenes  aureus  was 
found  in  the  vagina,  while  the  discharge  from  the  uterus  was  free 
from  germs.  In  five  cases  of  puerperal  fever  Streptococcus  pyogenes 
was  obtained  in  the  lochial  discharge  from  the  uterus.  The  results 
of  Yon  Ott  correspond  with  those  of  Donderlein.  Czerniewski,  in 
the  lochia  of  fifty-seven  healthy  women,  found  the  Streptococcus 
pyogenes  but  once,  while  in  the  lochial  discharge  of  fatal  cases  of 
puerperal  fever  it  was  always  present. 


AND  OF  EXPOSED  MUCOUS  MEMBRANES. 

Steffeck  (1892)  has  examined  the  vaginal  secretion  of  twenty-nine 
pregnant  females  who  had  not  been  subjected  to  digital  examina- 
tion, and  found  Staphylococcus  pyogenes  albus  in  nine,  Staphylo- 
coccus  pyogenes  aureus  in  three,  and  Streptococcus  pyogenes  in  one. 
These  results  indicate  that  puerperal  septicaemia  from  self-infection 
may  occur  in  exceptional  cases.  In  seventeen  of  the  twenty-nine 
cases  examined  none  of  these  pyogenic  micrococci  were  found. 

Hofmeister  (1894)  has  shown  that  bacteria  are  found  not  only 
upon  the  mucous  membrane  of  the  meatus  urinarius  in  man,  but 
that  they  may  usually  be  obtained  from  the  urethral  canal  at  a  depth 
of  eight  centimetres  or  more,  although  the  number  rapidly  diminishes 
in  the  deeper  portion  of  the  urethra. 

Walthard  (1895)  arrives  at  the  conclusion  that  while  in  pregnant 
females  bacteria  are  constantly  found  in  the  vagina  and  the  lower 
portion  of  the  cervical  canal,  they  are  absent  from  the  upper  part  of 
the  cervical  canal,  the  uterus,  and  the  tubes;  and  that  during  the 
puerperal  condition  the  uterine  cavity  is  preserved  from  spontaneous 
infection  per  vias  naturalis  by  the  plug  of  mucus  in  the  cervical 
canal.  In  the  vaginal  secretions  of  one  hundred  pregnant  women, 
who  had  not  been  subjected  to  a  digital  examination,  streptococci 
were  obtained  twenty-seven  times  in  cultures.  These  were  not  viru- 
lent, but,  according  to  Walthard,  these  saprophytic  streptococci  be- 
come virulent  when,  owing  to  a  diminished  resisting  power,  they  are 
enabled  to  invade  the  tissues  as  parasites. 

Kronig  (1894)  concludes  from  his  investigations  that  the  vaginal 
secretions  of  pregnant  women  are  usually  so  acid  that  Streptococcus 
pyogenes  could  not  multiply  in  them;  also  that  when  the  secretion  is 
normal  it  is  almost  always  sterile. 

Doclerlein  (1894)  insists  that  the  failure  of  Kronig  to  obtain  micro- 
organisms in  his  cultures  was  due  to  the  fact  that  suitable  media 
were  not  used ;  also  that  certain  bacilli  are  constantly  found  in  nor- 
mal, acid  vaginal  secretions,  and  that  in  the  pathological  secretions 
which  are  feebly  acid,  neutral,  or  in  some  cases  slightly  alkaline  a 
great  variety  of  bacteria  are  found,  including  Streptococcus  pyo- 
genes, as  demonstrated  by  himself  and  other  investigators.  In  a 
later  paper  (1894)  Kronig  reports  his  success  in  obtaining  cultures 
from  normal,  acid  vaginal  secretions  by  using  acid  media  and  by 
cultivating  under  anaerobic  conditions.  He  reports  also  that  patho- 
genic bacteria  (streptococci,  staphylococci,  and  Bacillus  pyocyaneus) 
introduced  into  the  vaginaB  of  pregnant  women  lose  their  power  of 
reproduction  in  from  six  to  forty-eight  hours  (streptococci  did  not 
grow  after  six  hours).  In  a  still  later  communication  (1894)  Kronig 
reports  that  the  bacteria  present  in  the  vaginal  secretions  of  pregnant 


656         BACTERIA  OF  THE  SURFACE  OF  THE  BODY 

women  are  for  the  most  part  strictly  anaerobic  species,  and  that 
among  these  he  found  two  non-pathogenic  streptococci. 

Menge  (189-4)  has  examined  the  vaginal  secretions  in  fifty  non- 
pregnant  women  who  had  been  in  bed  for  at  least  fourteen  days — 
after  laparotomy.  Microscopical  examination  showed  the  presence 
of  bacteria  in  all  cases,  but  in  only  six  cases  was  a  development  of 
colonies  obtained — upon  agar  plates;  in  one  case  Streptococcus  pyo- 
genes  was  present.  Menge  concludes  from  his  investigations  that 
spontaneous  infection  during  childbirth  cannot  occur,  and  that  with 
the  exception  of  the  gonococcus  the  known  pathogenic  bacteria  can- 
not multiply  in  the  cervical  canal. 

Gawronsky  (1894)  has  examined  the  secretions  from  the  healthy 
urethra  in  sixty-two  women,  most  of  whom  were  under  treatment 
for  uterine  disease  or  displacement.  The  material  for  his  cultures 
was  obtained  b}*  means  of  a  platinum  loop,  introduced  through  a 
glass  cylinder,  at  a  distance  of  one  or  one  and.  one-half  centimetres 
from  the  external  orifice  of  the  urethra.  In  fifteen  out  of  the  sixty- 
two  cases  examined  a  positive  result  was  obtained,  as  follows:  In 
three  cases  Streptococcus  pj^ogenes,  in  eight  Staphylococcus  pyogenes 
aureus,  in  one  Staphylococcus  pyogenes  albus,  in  two  Bacillus  coli 
communis,  in  one  Bacterium  tholoideum  of  Gessner. 

The  following  species  have  been  obtained  from  the  nasal  and 
buccal  secretions  : 

FROM   THE   NOSE. 

Non-pathogenic. — Micrococcus  nasalis  (Hajek),  Diplococcus  coryzoe 
(Hajek),  Micrococcus  albus  liquefaciens  (Von  Besser),  Micrococcus  cumu- 
latus  tenuis  (Von  Besser),  Micrococcus  tetragenus  subflavus  (Von  Besser), 
Diplococcus  fluorescens  f  retidus  (Klamann),  Micrococcus  foetidus  (Klamann), 
Vibrio  nasalis  (Weibel),  Bacillus  striatus  flavus  (Von.  Besser),  Bacillus 
striatus  albus  (Von  Besser). 

Pathogenic. — Staphylococcus  pyogenes  aureus,  Staphylococcus  pyogenes 
albus,  Streptococcus  pyogenes.  Bacillus  of  Friedlander,  Bacillus  of  rhino- 
scleroma  (?),  Bacillus  foetidus  ozaeiise  (Hajek),  Bacillus  mallei  (Loffler),  Ba- 
cillus smaragdmus  foetidus  (Reimann). 

FROM   THE   MOUTH. 

Non-pathogenic. — Micrococcus  roseus  (Eisenberg),  Micrococcus  A,  B,  C, 
D,  E  of  Podbielskij,  Sarcina  pulmonum  (Hauser),  Sarcina  lutea,  Micrococcus 
candicans  (Fliigge),  Bacillus  of  Miller,  Bacillus  virescens  (Frick),  Vibrio 
rugula,  Vibrio  lingualis  (Weibel),  Pseudo-diphtheria  bacillus  (Von  Hoff- 
mann), Bacillus  mesentericus  vulgatus,  Bacillus  subtiHs,  Bacillus  a,  b,  c,  d, 
e,  /,  gr,  h,  i,  and,/ of  Vignal,  Bacillus  subtilis  similis,  Bacillus  radiciformis 
(Eisenberg),  Bacillus  luteus,  Bacillus  fluorescens  non-liquefaciens,  Bacillus 
ruber,  Bacillus  viridiflavus,  Proteus  Zenkeri,  Bacillus  G,  H,  I,  J,  K,  L,  M. 
N,  and  Vibrio  O  andP  of  Podbielskij,  Vibrio  viridaiis  (Miller),  Micrococcus 
nexifer  (Miller),  lodococcus  magnus  (Miller),  Ascococcus  buccalis  (Miller), 
Bacillus  fuscans  (Miller). 

Pathogenic. — Staphylococcus  pyogenes  albus,  Staphylococcus  pyogenes 
aureus,  Staphylococcus  salivarius  septicus  (Biondi),  Streptococcus  pyogenes, 
Micrococcus  salivarius  septicus  (Biondi),  Micrococcus  tetra<jenus  (Gaffky), 


AXD    OF    EXPOSED    MUCOUS    MEMBRANES.  657 

Micrococcus  gingivaa  pyogenes  (Miller),  Streptococcus  septo-pyaeinicus  (Bi- 
ondi),  Streptococcus  articulorum  (Loffler),  Micrococcus  of  Manfredi,  Micro- 
coccus  pneuinonise  crouposae — ' '  Micrococcus  Pasteuri "  (Sternberg) ;  Bacillus 
diphtherias  (Loffler),  Bacillus  tuberculosis  (Koch),  Bacillus  of  Friedlander, 
Bacillus  bronchitidae  putridse  (Lurnnitzer),  Bacillus  septicaamiae  haemorrha- 
gicae,  Bacillus  gingivae  pyogenes  (Miller),  Bacillus  pulpse  pyogenes  (Miller), 
Bacillus  dentalis  viridans  (Miller),  Bacillus  crassus  sputigenus  (Kreibohm), 
Bacillus  saprogenes  No.  1  (Rosenbach),  Bacillus  pneumoniae  agilis  (Schou), 
Bacillus  pneumonias  of  Klein,  Bacillus  pneumosepticus  (Babes). 

42 


As  the  secretions  of  the  mouth  contain  numerous  bacteria,  these 
must  constantly  find  their  way  to  the  stomach,  but  conditions  are 
not  favorable  for  their  development  when  the  stomach  is  in  a  healthy 
state  and  its  secretions  normal.  Under  certain  circumstances,  how- 
ever, there  may  be  an  abundant  development  in  the  stomach  of  spe- 
cies which  give  rise  to  various  fermentations,  and  no  doubt  dyspep- 
tic symptoms  are  frequently  due  to  this  cause.  In  the  present 
section  we  are,  however,  only  concerned  with  the  bacteria  of  the 
healthy  stomach.  Most  of  these,  we  think,  are  to  be  considered  as 
only  temporarily  and  accidentally  present  in  this  viscus  as  the  result 
of  the  swallowing  of  the  buccal  secretions  and  of  food  and  drink  con- 
taining them. 

The  experiments  of  Straus  and  Wiirtz  and  of  others  show  that 
normal  gastric  juice  possesses  decided  germicidal  power,  which  is 
due  to  the  free  hydrochloric  acid  contained  in  it.  Hamburger  (1890) 
found  that  gastric  juice  containing  free  acid  is  almost  always  free 
from  living  microorganisms,  and  that  it  quickly  kills  the  cholera 
spirillum  and  the  typhoid  bacillus,  but  has  no  effect  upon  anthrax 
spores.  Straus  and  Wiirtz  found  that  the  cholera  spirillum  is  killed 
by  two  hours'  exposure  in  gastric  juice  obtained  from  dogs,  the 
typhoid  bacillus  in  two  to  three  hours,  anthrax  bacilli  in  fifteen  to 
twenty  minutes,  and  the  tubercle  bacillus  in  from  eighteen  to  thirty- 
six  hours.  The  experiments  of  Kurlow  and  Wagner,  made  with 
gastric  juice  obtained  from  the  stomach  of  healthy  men  by  means  of 
a  stomach  sound,  gave  the  following  results  :  Anthrax  bacilli  with- 
out spores  failed  to  grow  after  exposure  to  the  action  of  human  gas- 
tric juice  for  half  an  hour,  but  spores  were  not  destroyed  in  twenty- 
four  hours ;  the  typhoid  bacillus  was  killed  in  one  hour ;  the 
cholera  spirillum,  the  bacillus  of  glanders,  and  Bacillus  pyocyanus 
were  all  destroyed  at  the  end  of  half  an  hour  ;  the  pus  cocci  showed 
greater  resisting  power.  Certain  bacteria  have  a  greater  resisting 
power  for  acids  than  any  of  those  above  mentioned,  and  some  of  them 
may  consequently  pass  through  the  healthy  stomach  to  the  intestine 


BACTERIA    OF    THE    STOMACH    AXD    INTESTINE.  659 

in  a  living  condition,  but  there  is  good  reason  to  believe  that  t_ie 
spirillum  of  cholera  or  the  bacillus  of  anthrax  would  not.  On  tli3 
other  hand,  the  tubercle  bacillus  and  the  spores  of  other  bacilli  can, 
no  doubt,  pass  through  the  stomach  to  the  intestine  without  losing 
their  vitality. 

Of  nineteen  species  isolated  by  Vignal  in  his  cultures  from  the 
healthy  human  mouth,  the  greater  number  resisted  the  action  of  the 
gastric  juice  for  more  than  an  hour,  and  six  species  which  did  not 
form  spores  were  found  to  retain  their  vitality  in  gastric  juice  for 
more  than  twenty-four  hours. 

In  making  a  bacteriological  analysis  of  the  contents  of  the  healthy 
stomach  the  more  resistant  microorganisms  and  those  which  form 
spores  will. naturally  be  found  in  greater  or  less  numbers,  inasmuch 
as  some  of  them  are  likely  to  be  present  in  food  and  water  ingested. 

Yan  Puteren  (1888)  obtained  a  variety  of  microorganisms  in  very 
considerable  numbers  from  the  stomachs  of  infants  fed  upon  un- 
sterilized  cow's  milk,  but  in  healthy  nursing  infants  the  number  was 
much  smaller,  especially  when  the  mouth  was  washed  out  with  dis- 
tilled water  immediately  before  and  after  nursing.  In  18  per  cent 
of  the  cases  no  microorganisms  were  found  under  these  circum- 
stances, and  in  41  per  cent  the  number  fell  below  one  thousand  per 
cubic  centimetre,  Among  the  nursing  infants  examined  (eighty- 
five)  the  following  species  were  most  numerous  :  Monilia  Candida, 
Bacillus  lactis  aerogenes,  a  non-liquefying  coccus,  Staphylococcus 
pyogenes  aureus,  Bacillus  subtilis.  In  infants  fed  upon  cow's  milk 
(eleven)  Bacillus  lactis  aerogenes  was  present  in  45.4  per  cent  of 
the  cases,  and  Staphylococcus  pyogenes  aureus  in  27.2  per  cent,  non- 
liquefying  cocci  in  54. 4  per  cent,  liquefying  cocci  in  72. 7  per  cent, 
Bacillus  subtilis  in  36.3  percent,  and  Bacillus  butyricus  (Hueppe) 
in  all  of  the  cases  ;  next  to  these  Bacillus  flavescens  liquefaciens 
was  the  most  abundant.  The  author  named  reaches  the  conclusion 
that  no  species  is  constant  and  that  the  presence  of  those  found  de- 
pends upon  accidental  circumstances. 

Abelous  (1889)  found  in  his  own  stomach,  washed  out  while  fast- 
ing, a  considerable  number  of  species  of  bacteria,  viz*  :  Sarcina 
ventriculi,  Bacillus  pyocyaneus,  Bacillus  lactis  aerogenes,  Bacillus 
subtilis,  Bacillus  mycoides,  Bacillus  amylobacter,  Vibrio  rugula, 
and  eight  other  undescribed  bacilli  and  one  coccus.  All  of  these 
microorganisms  were  able  to  resist  the  action  of  hydrochloric  acid 
in  the  proportion  of  1.7  grammes  in  1,000  grammes  of  water. 
Several  were  found  to  be  facultative  anaerobics. 

The  action  of  the  bacteria  isolated  by  him  was  tested  by  Abelous 
upon  various  alimentary  substances.  The  time  required  to  effect 
changes,  such  as  the  digestion  of  fibrin,  the  changing  of  starch 


GGO  BACTERIA    OF    THE    STOMACH   AND   INTESTINE. 

into  glucose,  etc,,  was  found  to  be  so  long  that  there  was  no  reason 
to  suppose  that  any  one  of  the  microorganisms  tested  was  con- 
cerned in  ordinary  stomach  digestion. 

In  the  intestine  conditions  are  favorable  for  the  development  of 
many  species  of  saprophytie  bacteria,  and  the  smallest  quantity  of 
excrementitious  material  from  the  bowels,  spread  upon  a  glass  slide 
and  stained  with  one  of  the  aniline  colors,  will  be  found  to  contain 
a  multitude  of  microorganisms  of  this  class,  of  various  forms. 
Among  these  are  certain  species  which  have  their  normal  habitat  in 
the  intestine,  and  which  may  always  be  obtained  in  cultures  from 
this  source,  while  others,  having  been  present  in  food  or  water  in- 
gested, and  having  escaped  destruction  in  the  acid  juices  of  the 
stomach,  are  accidentally  and  temporarily  present.  These  latter 
may  or  may  not  increase  in  the  organic  pabulum  which  abounds  in 
the  intestine,  according  as  the  conditions  are  favorable  or  otherwise. 
The  strictly  aerobic  bacteria  could  not  multiply  because  of  the  ab- 
sence of  oxygen,  and  the  species  encountered  are  for  the  most  part 
anaerobics  or  facultative  anaerobics.  The  Bacillus  coli  communis 
of  Escherich,  which  is  the  most  constant  and  abundant  species  found 
in  the  intestine  of  man  and  of  certain  of  the  lower  animals,  is  a  facul- 
tative anaerobic,  which  grows  readily  in  the  ordinary  culture  media, 
either  in  the  presence  of  oxygen  or  in  an  atmosphere  of  hydrogen. 
But  certain  other  bacteria  of  the  intestine  are  strictly  anaerobic  and 
do  not  grow  readily  in  the  media  commonly  employed  by  bacteri- 
ologists. 

Escherich  has  shown  that  in  new-born  infants  the  meconium  is 
free  from  bacteria.  At  the  end  of  twelve  to  eighteen  hours  after 
birth  bacteria  appear  in  the  alvine  discharges,  and  the  number  is 
already  considerable  at  the  expiration  of  the  first  twenty-four  hours 
of  independent  existence.  The  species  first  found  are  cocci  and  yeast 
cells  which  no  doubt  come  from  the  atmosphere,  having  been  de- 
posited upon  the  moist  mucous  membrane  of  the  mouth  and  swal- 
lowed with  the  buccal  secretions.  When  the  meconium  is  replaced 
by  "  milk  faeces "  these  contain  in  large  numbers  the  Bacillus  coli 
communis,  heretofore  spoken  of  as  the  most  common  species  found  in 
the  intestine  of  adults.  Another  species  associated  with  this,  but 
not  so  abundant,  is  the  Bacillus  lactis  aerogenes  of  Escherich. 
Other  bacilli  and  cocci  are  found  occasionally  in  smaller  numbers. 
These  bacilli  do  pot  liquefy  gelatin,  and,  as  a  rule,  the  microor- 
ganisms found  in  the  alvine  discharges  of  healthy  persons  are  noii- 
liquefying  bacteria.  Eschericlrs  researches  led  him  to  the  conclu- 
sion that  the  Bacillus  lactis  aerogenes  is  constantly  present  in  the 
small  intestine  of  milk-fed  children  as  the  most  prominent  species, 
and  that  its  multiplication  there  is  favored  by  the  presence  of  milk 


BACTERIA   OF   THE   STOMACH   AND    INTESTINE.  661 

sugar,   and  that  Bacillus   coli  communis  finds  the  most  favorable 
conditions  for  its  growth  in  the  large  intestine. 

Brieger,  in  1884,  isolated  from  faeces  and  carefully  studied  two 
bacilli,  one  of  which  has  since  been  called  by  his  name.  This  is  a 
non-liquefying  bacillus  which  is  very  pathogenic  for  guinea-pigs, 
and  which  in  its  morphology  and  characters  of  growth  closely  re- 
sembles the  Bacillus  coli  communis  of  Escherich.  Indeed,  a  num- 
ber of  non-liquefying  bacilli,  differing  but  slightly  in  their  morpho- 
logical and  biological  characters,  have  been  obtained  by  various 
investigators  from  the  alimentary  canal  of  man  and  the  lower  ani- 
mals, and  it  is  still  a  question  whether  they  are  to  be  regarded  as 
distinct  species  or  as  varieties  of  the  "colon  bacillus  "  of  Escherich. 
The  bacillus  obtained  by  Emmerich  from  cholera  cadavers  in  Na- 
ples belongs  to  this  group,  and,  if  not  identical  with  the  colon  bacil- 
lus, resembles  it  so  closely  that  its  differentiation  is  extremely  diffi- 
cult. Brieger's  bacillus  forms  propionic  acid  in  solutions  containing, 
grape  sugar.  A  second  bacillus  obtained  by  him  from  the  same 
source  resembles  the  * '  pneumococcus  "  of  Friedlander  ;  this  causes 
the  fermentation  of  saccharine  solutions,  with  production  of  ethyl 
alcohol. 

Bienstock  (1883)  isolated  four  species  of  bacilli  from  normal  faeces, 
two  of  which  are  comparatively  large  and  resemble  Bacillus  sub- 
tilis  in  their  morphology  and  in  the  formation  of  spores.  A  third 
species  is  described  as  an  extremely  slender  pathogenic  bacillus,  re- 
sembling the  bacillus  of  mouse  septicaemia.  The  fourth  species  is  an 
actively  motile  bacillus  which  forms  end  spores,  causing  the  rods  to 
have  the  form  of  a  drumstick.  This  is  said  to  cause  the  decomposi- 
tion of  albumin,  with  production  of  ammonia  and  carbon  dioxide. 
Later  researches  do  not  sustain  Bienstock's  conclusion  that  the  ba- 
cilli described  by  him  are  the  principal  forms  found  in  normal  faeces. 

Among  the  species  encountered  by  Escherich,  in  addition  to  those 
mentioned  above  (Bacillus  coli  communis  and  Bacillus  lactis  aero- 
genes),  are  the  following  :  Proteus  vulgaris,  found  three  times  in 
meconium,  and  constantly  in  the  faeces  of  dogs  fed  upon  flesh  ;  Strep- 
tococcus coli  gracilis,  found  in  meconium,  but  not  during  the  period 
of  nursing,  is  constantly  present  in  the  intestine  when  a  flesh  diet  is 
employed. 

The  intestine  of  carnivorous  and  omnivorous  animals  contains  a 
greater  number  of  bacteria  than  that  of  the  herbivora,  and  in  the 
large  intestine  they  are  far  more  numerous  than  in  the  small  intes- 
tine (De  Giaxa).  Sucksdorf  has  enumerated  the  colonies  developing 
from  one  milligramme  of  faeces  from  individuals  on  mixed  diet.  He 
obtained  an  average  of  380,000  from  a  series  of  observations  in  which 
the  maximum  was  2,300,000  and  the  minimum  25,000. 


CG2  BACTERIA    OF    THE    STOMACH   AND    INTESTINE. 

The  constant  presence  of  certain  species  of  bacteria  in  the  intes- 
tine of  man  and  the  lower  animals  has  led  to  the  supposition  that 
they  may  serve  a  useful  purpose,  or  perhaps  even  have  an  essential 
physiological  role  in  connection  with  intestinal  digestion.  While 
this  question  has  not  been  definitely  settled,  the  experiments  of 
Vallin,  Abelous,  and  others  have  thrown  some  light  upon  it,  and  a 
recent  experiment  by  Nuttall  and  Thierf elder  (1895)  has  considerable 
importance  as  bearing  upon  its  solution.  The  experiment  consisted 
in  removing  a  foetus  from  a  pregnant  guinea-pig  by  Caesarean  sec- 
tion, placing  it  under  conditions  which  protected  it  from  the  micro- 
organisms present  in  the  atmosphere,  and  feeding  it  upon  sterilized 
milk.  Great  technical  skill  was  shown  in  carrying  out  this  experi- 
ment for  a  period  of  eight  days,  during  which  time  the  little  animal 
was  kept  in  a  sterilized  atmosphere  and  was  fed  every  hour  day  and 
night.  At  the  end  of  this  time  it  had  consumed  over  three  hundred 
and  thirty  cubic  centimetres  of  sterilized  milk,  and  was  as  active  and 
healthy  as  other  guinea-pigs  of  the  same  age.  It  was  now  killed,  and 
a  careful  bacteriological  examination  showed  that  the  discharges 
from  the  bowels  and  the  contents  of  the  intestine  were  entirely  sterile. 

ADDITIONAL   NOTES   UPON   BACTERIA  OF    THE    STOMACH   AND 

INTESTINE. 

Oppler  (1894)  has  examined  material,  obtained  in  the  early  morning,  from 
the  stomach  of  persons  suffering  from  indigestion,  and  found  nearly  always 
numerous  masses  of  sarcinae.  Five  different  species  were  obtained  from  this 
source,  which  were  distinguished  by  the  following  characters  :  No.  1,  colo- 
nies sulphur  yellow ;  No.  2,  colonies  greenish  yellow  ;  No.  3,  colonies  white ; 
No.  4,  colonies  white,  does  not  liquefy  gelatin  ;  No.  5,  colonies  orange  yel- 
low. Nos.  1  and  3  were  most  frequently  encountered. 

Kauffmann  (1895)  in  a  carefully  studied  case  of  chronic  dyspepsia  obtained 
from  the  contents  of  the  stomach  in  the  morning  before  breakfast,  and  after 
a  test  meal,  the  following  bacteria  :  Yellow  sarcina,  Micrococcusaurantiacus, 
Staphylococcus  cereus  albus.  Bacillus  subtilis,  Bacillus  ramosus,  ' '  a  large 
thick  bacillus,"  "  a  short  bacillus  resembling  Bacillus  coli  communis."  The 
last-mentioned  bacillus  was  found  in  large  numbers,  and  Kauffmami  suggests 
that  it  may  have  been  the  cause  of  the  fermentation  in  the  digestive  tract 
which  caused  the  unpleasant  symptoms  in  the  case  under  investigation. 

Macf ayden  (1887)  and  Gillespie  (1893)  have  also  obtained  a  bacillus  from 
the  stomach  which  appears  to  be  identical  with  Bacillus  coli  communis.  In 
the  researches  of  Gillespie  it  was  obtained  from  a  patient  with  dilatation  of 
the  stomach  who  suffered  from  flatulence,  etc.  In  all,  twenty-four  different 
microorganisms  were  obtained  by  Gillespie  from  the  contents  of  the  stomach 
of  different  individuals.  This  number  includes  three  species  of  saccha- 
romyces  and  a  mucor.  Among  the  conclusions  reached  by  Gillespie  are  the 
following  : 

"14.  Although  bacteria  are  of  no  aid  to  peptic  digestion,  and  a  hindrance 
to  the  pancreatic  ferment  if  in  quantity  in  the  duodenum,  they  still  are  of 
great  use  in  the  small  intestine,  where  they  control  putrefaction.  This  seems 
paradoxical  :  microorganisms  obstructing  microorganisms  but  assisting  diges- 
tion. It  seems,  however,  to  be  true.  The  organisms  which  most  easily 
pass  the  searching  examination  of  the  stomach  are  those  which  give  rise  by 


BACTERIA   OF   THE   STOMACH   AND   INTESTINE.  663 

their  growth  to  the  fatty  acids,  as  they  are  the  most  resistant  to  the  action  of 
acids.  Their  products  in  the  small  intestine  are  sufficient  to  keep  the  con- 
tents of  that  viscus  acid,  and  they  thereby  prevent  or  control  putrefaction. 
In  the  large  intestine  the  secretion  is  so  alkaline  that  the  putrefactive  organ- 
isms reassert  themselves. 

"15.  Increased  putrefaction  in  the  intestinal  canal  may  therefore  be  due, 
in  some  cases,  either  to  insufficient  mortality  among  the  putrefactive  organ- 
isms in  the  stomach,  or  to  too  great  mortality  among  the  acid-forming  bac- 
teria and  yeasts. 

"16.  The  lactic  acid  which  appears  during  the  first  stages  of  digestion  is 
due  to  the  action  of  organisms. 

"17.  The  lactic,  acetic,  butyric,  and  succiiiic  acids  found  in  gastroectasis 
are  due  also  to  organisms  which  luxuriate  in  the  too  stationary  contents. 
The  marsh  gas,  the  Brennender-gas  of  the  Germans,  is  probably  due  to  the 
same  cause  ;  in  the  only  case  of  this  character  with  which  I  have  had  the 
good  fortune  to  meet  no  material  for  examination  could  be  obtained." 

The  following  species  have  been  isolated  from  faeces  and  the  con- 
tents of  the  intestine  of  cadavers  : 

Non-pathogenic. — Streptococcus  coli  gracilis  (Escherich),  Micrococcus 
aerogenes  (Miller),  Micrococcus  tetragenus  versatilis  (Sternberg),  Micrococ- 
cus ovalis  (Escherich),  "Yellow  liquefying  staphylococcus"  (Escherich), 
"  Porzellancoccus  "  (Escherich),  Bacillus  subtilis,  Bacillus  aerogenes  (Miller), 
Bacterium  aerogenes  (Miller),  Bacillus lactis erythrogenes  (Hueppe),  Clostri- 
dium  fcetidum  (Liborius),  Bacillus  muscoides  (Liborius) ,  Bacillus  putrificus 
coli  (Bienstock),  Bacillus  subtilis  similis  I.  and  II.  (Bienstock),  Bacillus 
Zopfii,  Bacillus  liquefaciens  communis  (Sternberg),  Bacillus  intestinus  lique- 
faciens  (Sternberg),  Bacillus  intestinus  motilis  (Sternberg),  Bacillus  fluores- 
cens  liquefaciens  (Fliig-ge),  "Colorless  fluorescent  liquefying  bacillus" 
(Escherich),  "Yellow  liquefying  bacillus"  (Escherich),  Bacillus  mesenteri- 
cus  vulgatus,  Bacilli  of  Booker,  A  to  T,  first  series ;  a  to  s,  second  series ; 
Bacilli  of  Jeffries  A  to  Z,  and  or,  ft. 

^  Pathogenic.—  Staphylococcus  pyogenes  aureus,  Bacillus  typhi  abdo- 
minalis,  Bacillus  septicaemise  haemorrhagicaa,  Bacillus  of  Belfanti  and  Pas- 
carola,  Bacillus  enteritidis  (Gartner),  Bacillus  of  Lesage,  Bacillus  pseudo- 
murisepticus  (Bienstock),  Bacillus  coli  communis  (Escherich),  Bacillus  lactis 
aerogenes  (Escherich),  Bacillus  cavicida  (Brieger),  Bacillus  of  Emmerich, 
Bacillus  coprogenes  fcetidus  (Schottelius),  Bacillus  of  Utpadel,  Bacillus  leporis 
lethalis  (Sternberg),  Bacillus  acidiformans  (Sternberg),  Bacillus  cuniculicida 
Havaniensis  (Sternberg),  Bacillus  cadaveris  (Sternberg),  Bacillus  cavicida 
Havaniensis  (Sternberg),  Proteus  vulgaris  (Hauser),  Bacillus  tuberculosis, 
Spirillum  choleras  Asiaticee  Spirillum  of  Finkler  and  Prior. 


VI. 

BACTERIA  OF    CADAVERS   AND    OF    PUTREFYING 
MATERIAL  FROM  VARIOUS  SOURCES. 

THE  putrefactive  changes  which  occur  so  promptly  in  cadavers, 
when  temperature  conditions  are  favorable,  result  chiefly  from  post- 
mortem invasion  of  the  tissues  by  bacteria  contained  in  the  alimen- 
tary canal.  But  it  is  probable  that  under  certain  circumstances 
microorganisms  from  the  intestine  may  find  their  way  into  the  cir- 
culation during  the  last  hours  of  life,  and  that  the  very  prompt  putre- 
factive changes  in  certain  infectious  diseases  in  which  the  intestine 
is  more  or  less  involved  are  due  to  this  fact.  The  writer  has  made 
numerous  experiments  in  which  a  portion  of  liver  or  kidney  re- 
moved from  the  cadaver  at  an  autopsy  made  soon  after  death — one 
to  six  hours — has  been  enveloped  in  an  antiseptic  wrapping  and  kept 
for  forty-eight  hours  at  a  temperature  of  25°  to  30°  C.  In  every  in- 
stance there  has  been  an  abundant  development  of  bacteria,  although 
as  a  rule  none  were  obtained  from  the  same  material  immediately  after 
the  removal  of  the  organ  from  the  body.  This  shows  that  a  few 
scattered  bacteria  were  present.  The  same  result  was  obtained  in 
cases  of  sudden  death  from  accident,  as  from  portions  of  liver  or 
kidney  removed  from  the  bodies  of  persons  dying  of  yellow  fever, 
tuberculosis,  and  other  diseases. 

Numerous  researches  show  that  the  blood  of  healthy  men  and 
animals  is  free  from  bacteria,  and  that  saprophytic  bacteria  injected 
into  a  vein  soon  disappear  from  the  circulation  ;  and  recent  experi- 
ments show  that  blood  serum  has  decided  germicidal  power.  But  in 
spite  of  this  fact  the  experiments  of  Wyssokowitsch  show  that  cer- 
tain bacteria  injected  into  the  circulation  may  be  deposited  in  the 
liver,  the  spleen,  and  the  marrow  of  the  bones,  and  there  retain  their 
vitality  for  a  considerable  time.  The  spores  of  Bacillus  subtilis  were 
found  by  the  observer  named  to  preserve  their  vitality  in  the  liver  or 
spleen  of  animals  into  which  they  had  been  injected,  for  a  period  of 
two  or  three  months.  In  the  writer's  experiments  the  microorgan- 
isms which  first  developed  in  fragments  of  liver  preserved  in  an  an- 
tiseptic wrapping  were  certain  large  anaerobic  bacilli,  and  especially 


BACTERIA   OF   CADAVERS   AND    OF   PUTREFYING   MATERIAL.       C05 


my  Bacillus  cadaveris,  together  with  the  Bacillus  coli  communis 
of  Escherich,  my  Bacillus  hepaticus  fortuitus,  and  other  non-lique- 
fying bacilli  of  the  "  colon  group/'7 

These  bacteria  did  not  give  rise  to  a  putrefactive  odor,  and  the 
fragment  of  liver  when  cut  into  had  a  fresh  appearance  and  a  very 
acid  reaction.  Later,  putrefactive  changes  occurred  and  Proteus 


FIG.  197.—  Smear  preparation  from  liver  of  yellow-fever  cadaver,  kept  forty-eight  hours  in  an 
antiseptic  wrapping,    x  1,000.    From  a  photomicrograph.     (Steinberg.) 

vulgaris  and  other  putrefactive  bacteria  obtained  the  precedence. 
Evidently  all  of  these  species  must  have  been  present  in  the  liver  at 
the  time  it  was  removed  from  the  cadaver,  although  in  such  small 
numbers  that  they  were  rarely  seen  in  smear  preparations  or  ob- 
tained in  cultures  from  the  fresh  liver  tissue.  The  appearance  of  a 
smear  preparation  from  the  interior  of  a  fragment  preserved  for 
forty-eight  hours  in  an  antiseptic  wrapping  is  shown  in  Fig.  197. 

The  horribly  offensive  gases  which  are  given  off  from  dead  ani- 
mals in  a  state  of  putrefaction  appear  to  be  due  to  certain  large  an- 
aerobic bacilli  which  are  found  in  such  material, 
and  which  have  not  yet  been  thoroughly  studied  ^k 

owing  to  the  difficulty  of  cultivating  them  in  arti- 
ficial  media  ;  among  them  is  a  large  bacillus  with 
round  ends  which  forms  an  oval  spore  at  one  ex- 
tremity  of  the  rather  long  rod.  This  the  writer 
has  described  under  the  name  of  Bacillus  cada- 
veris  grandis,  Fig.  198. 

In  the  interior  of  a  putrefying  mass  of  this  kind 
only  those  bacteria  are  found  which  are  able  to  grow  in  the  absence 
of  oxygen,  but  aerobic  saprophytes  may  multiply  upon  the  surface  of 


FIG 


666      BACTERIA   OF   CADAVERS   AND   OF   PUTREFYING  MATERIAL. 

such  a  mass,  or  in  organic  liquids  to  which  the  air  has  free  access. 
Among  the  most  common  putrefactive  bacteria  are  the  Proteus  vul- 
garis,  Proteus  mirabilis,  and  Proteus  Zenkeri  of  Hauser.  Formerly 
the  minute  motile  bacteria  found  in  putrefying  animal  infusions,  etc., 
were  commonly  spoken  of  as  belonging  to  the  species  "  Bacterium 
termo,"  but  recent  researches  show  that  several  different  species  were 
included  under  this  name  by  those  whose  researches  were  made  be- 
fore the  introduction  of  Koch's  method  for  isolating  and  differentiat- 
ing microorganisms  of  this  class  by  the  use  of  solid  culture  media. 
The  different  species  of  Proteus  are  all  facultative  anaerobics.  They 
are  more  or  less  pathogenic,  and  according  to  Hauser  produce  a  chem- 
ical poison  which,  when  injected  into  small  animals,  causes  death  with 
all  of  the  symptoms  of  putrid  intoxication.  The  bacillus  of  mouse 
septicaemia,  which  was  first  obtained  by  Koch  from  a  putrefying  meat 
infusion,  is  also  pathogenic,  as  are  the  writer's  Bacillus  cadaveris 
and  various  other  anaerobic  bacteria  found  in  putrefying  material. 

Some  account  of  the  various  products  of  putrefaction  and  the 
microorganisms  concerned  in  their  production  will  be  found  in  Sec- 
tion IV.,  Part  Second,  of  the  present  volume. 


VII. 

BACTERIA  IN  ARTICLES  OF  FOOD. 

Milk  always  contains  bacteria,  unless  drawn  with  special  precau- 
tions into  a  sterilized  flask.  In  the  healthy  udder  of  the  cow  it  is 
sterile,  but  in  tuberculous  cows,  when  the  milk  glands  are  involved, 
tubercle  bacilli  may  find  their  way  into  the  milk  in  considerable 
numbers.  Ag  ordinarily  obtained  and  preserved,  milk  is  greatly  ex- 
posed to  bacterial  contamination  from  various  sources  ;  desquamated 
cuticle  from  the  external  surface  of  the  udder  and  from  the  hands  of 
the  milker,  and  floating  particles  from  the  air  of  the  stable,  fall  into  it 
at  the  very  moment  it  is  drawn,  and  it  is  subsequently  contaminated 
by  bacteria  from  the  air,  and  from  water  used  in  washing  the  recep- 
tacles in  which  it  is  placed  or  added  to  it  by  the  thrifty  milkman. 
As  it  furnishes  an  excellent  nutrient  medium  for  many  of  the  bacteria 
which  are  thus  introduced  into  it,  under  favorable  conditions  of  tem- 
perature it  quickly  undergoes  changes  due  to  the  multiplication  in  it 
of  one  or  more  of  these  microorganisms.  The  acid  fermentation  and 
coagulation  of  the  casein  which  so  constantly  occurs  is  completely 
prevented  by  sterilizing  fresh  milk  in  flasks  provided  with  a  close- 
fitting  cork  or  cotton  air  filter.  Numerous  researches  have  been 
made  with  reference  to  the  microorganisms  found  in  milk  and  the 
various  fermentations  to  which  they  give  rise.  Naturally  a  great 
variety  of  species  will  be  found  in  an  extended  research,  but  all  are 
accidentally  present,  and  only  those  demand  special  attention  which 
produce  the  various  fermentations  of  this  fluid  commonly  encoun- 
tered, or  which  have  special  pathogenic  properties. 

Several  different  bacteria  produce  an  acid  fermentation  and  con- 
sequent coagulation  of  milk,  but  the  usual  agent  in  producing  this 
fermentation  is  the  Bacillus  acidi  lactici,  which  is  identical  with  the 
' '  ferment  lactique  "  of  Pasteur.  When  a  pure  culture  of  this  bacillus 
is  introduced  into  sterilized  milk  kept  at  a  temperature  of  25°  to  30°  C., 
coagulation  occurs  in  from  fifteen  to  twenty-four  hours.  A  uniform, 
gelatinous  mass  is  produced  which  does  not  subsequently  become 
dissolved  (Adametz).  Various  other  bacteria  produce  a  similar 
change,  including  a  number  of  common  water  bacteria,  several  spe- 


668  15. UTERI  A   IN   ARTICLES   OF   FOOD. 

cies  of  sarcina,  Staphylococcus  pyogenes  aureus,  and  other  pus  cocci. 
Usually  coagulation  is  due  to  the  combined  action  of  several  bacteria, 
among  which  Bacillus  acidi  lactici  is  apt  to  be  the  most  prominent. 

Other  bacteria  produce  coagulation  without  the  lactic  acid  fer- 
mentation. This  appears  to  be  due  to  the  formation  of  a  soluble 
ferment  which  acts  like  rennet,  causing  the  coagulation  of  milk 
which  has  a  neutral  or  slightly  alkaline  reaction.  The  coagu- 
lated casein  in  this  case  is  subsequently  redissolved.  The  bacteria 
which  produce  this  change  for  the  most  part  form  spores,  while  the 
lactic  acid  ferments  do  not.  If,  therefore,  milk  is  heated  nearly  to  the 
boiling  point  the  acid-forming  bacteria  will  be  destroyed  and  the 
spores  of  the  other  species  surviving  will  give  rise  to  coagulation 
without  the  production  of  lactic  acid.  Among  the  more  common 
microorganisms  of  this  group  are  the  Bacillus  butyricus  (Hueppe), 
Bacillus  mesentericus  vulgatus,  Loffler's  "  white  milk-bacillus,"  and 
the  bacilli  described  by  Duclaux  under  the  generic  name  of  Tyrothrix. 

Other  fermentations  are  produced  by  certain  chromogenic  bacteria, 
and  these,  as  a  rule,  are  not  as  harmless  from  a  sanitary  point  of  view 
as  those  above  referred  to.  Blue  milk  is  produced  by  the  presence  of 
Bacillus  cyanogenus,  yellow  milk  by  Bacillus  synxanthus  (Schroter) 
and  by  a  species  obtained  by  List  from  the  faeces  of  a  sheep  and 
another  found  by  Adametz  in  cheese.  The  well-known  Bacillus 
prodigiosus  produces  its  characteristic  red  pigment  when  present  in 
milk,  and  a  bluish-red  color  is  caused  by  Bacterium  lactis  ery throgenes 
(Hueppe). 

Viscous  fermentation  in  milk  is  produced  by  several  different  bac- 
teria, among  others  by  a  micrococcus  studied  by  Schmidt-Muhlheim, 
and  a  short  bacillus  isolated  by  Adametz — Bacillus  lactis  viscosus. 
Milk  which  has  undergone  this  change  is  unwholesome  as  food  ;  it 
is  recognized  by  the  long  filaments  which  are  produced  when  it  is 
touched  with  any  object  and  this  is  slowly  withdrawn. 

The  Caucasian  milk  ferment,  Bacillus  Caucasicus,  produces  a 
special  fermentation,  which  has  been  referred  to  in  Section  IV. ,  Part 
Second  (page  139). 

Various  pathogenic  bacteria  have  occasionally  been  found  in  milk 
in  addition  to  the  tubercle  bacillus  already  referred  to.  Thus  Adametz 
found  Staphylococcus  pyogenes  aureus  in  two  samples  which  had 
been  submitted  to  him  for  examination,  one  of  which  had  given  rise 
to  vomiting  and  diarrhoea.  Wyssokowitsch  cultivated  from  milk 
which  had  been  standing  some  time  a  pathogenic  bacillus,  named  by 
him  Bacillus  oxytocus  perniciosus. 

The  special  microorganism  which  produces  the  poisonous  pto- 
maine called  by  Vaughan  tyrotoxicon  has  not  yet  been  isolated  ;  nor 
do  we  know  the  exact  cause  of  scarlet  fever,  although  there  is  evi- 


BACTERIA    IX   ARTICLES    OF   FOOD. 


669 


dence  that  this  disease  lias  been  spread  by  the  use  of  contaminated 
milk,  as  have  also  diphtheria  and  typhoid  fever,  which  diseases  are 
due  to  bacilli  now  well  known.  As  the  cholera  spirillum  grows 
readily  in  milk,  this  disease  could  no  doubt  also  be  transmitted  in 
the  same  way. 

Sedgwick  and  Batchelder  (1892)  have  examined  a  large  number 
of  specimens  of  milk  obtained  in  Boston  and  vicinity,  for  the  purpose 
of  determining  the  number  of  bacteria  present.  They  found,  as  an 
average  of  several  trials,  that  milk  obtained  in  a  clean  stable,  from 
a  well-kept  cow,  by  milking  in  the  usual  way  into  a  sterilized  bottle, 
contained  530  bacteria  per  cubic  centimetre.  'When,  however,  the 
milkman  used  the  ordinary  milk  pail  of  flaring  form,  seated  himself 
with  more  or  less  disturbance  of  the  bedding,  and  vigorously  shook 
the  udder  over  the  pail  during  the  usual  process  of  milking,*'  the 
numbers  were  very  much  higher — on  an  average  30,500  per  cubic 
centimetre  immediately  after  milking.  The  average  of  fifteen  sam- 
ples taken  from  the  tables  of  persons  living  in  the  suburbs  of  Boston 
was  09,143  per  cubic  centimetre.  The  average  of  fifty-seven  sam- 
ples of  Boston  milk,  obtained  directly  from  the  milk  wagons  and 
plated  at  once,  was  2,355,500  per  cubic  centimetre.  The  average  of 
sixteen  samples  from  groceries  in  the  city  of  Boston  was  4,577,000 
per  cubic  centimetre. 

Prof.  Eenk  found  in  the  milk  supply  of  Halle  from  6,000,000  to 
oU, 000, 000  bacteria  per  cubic  centimetre — a  number  considerably  ex- 
ceeding that  usually  found  in  the  sewage  of  American  cities  (Sedg- 
wick). 

Cohn  and  Neumann  (1801)  have  shown  that  the  milk  of  healthy 
women  frequently  contains  bacteria,  and  that  Staphylococcus  pyo- 
genes  albus  is  the  species  most  frequently  found.  This  has  been 
confirmed  by  the  researches  of  Palleske  (1892),  Ringel  (1893)  and 
others.  The  last-mentioned  author  examined  the  milk  of  25  women 
recently  confined,  "12  of  whom  were  healthy  and  13  sick."  In  3 
cases  only  was  the  milk  sterile;  in  17  cases  Staphylococcus  pyogenes 
albus  was  found;  in  2  cases  Staphylococcus  pyogenes  aureus;  in  1 
case  both  albus  and  aurens ;  in  2  cases  Staphylococcus  pyogenes  albus 
and  Streptococcus  pyogenes.  The  streptococci  were  found  in  a  case 
of  mild  puerperal  fever  and  in  a  case  of  phlebitis. 

The  researches  of  Hirshberger  (1889),  of  Ernst  (1895),  and  of 
others  show  that  the  milk  of  tuberculous  cows  may  contain  tubercle 
bacilli  even  when  the  udder  of  the  animal  presents  no  evidence  of  a 
localized  tubercular  infection.  In  121  samples  of  milk  examined  by 
Ernst  from  36  different  cows,  19  gave  a  positive  result;  all  from  the 
milk  of  12  cows  in  which  no  evidence  of  tuberculosis  of  the  udder 
was  found  in  a  carefully  made  post-mortem  examination.  Among 


670  BACTERIA   IN  ARTICLES   OP  FOOD. 

the  bacteria  which  produce  unwholesome  chaL-g^s  in  milk  are  several 
which  cause  it  to  become  viscous  or  soapy.  Among  these  we  may 
mention  Micrococcus  lactis  viscosus  of  Conn,  Micrococcus  Freuden- 
reichi  of  Guillebeau,  Bacillus  mesentericus  vulgatus,  and  Bacillus 
lactis  saponacei  of  Weighmann  and  Zirn.  A  considerable  number 
of  bacilli  are  known  which  give  rise  to  the  production  of  butyric 
acid  fermentation  in  milk  and  its  products.  Some  of  these  are  an- 
aerobic and  some  aerobic.  The  list  includes  the  following :  Bacillus 
butyricus  of  Prazmowski,  Bacillus  of  Liborius,  Bacillus  of  Botkin, 
Bacilli  of  Kadrowski. 

The  bitter  taste  which  milk  and  cheese  sometimes  acquire  is  due 
to  the  presence  of  special  bacterial  ferments ;  among  these  the  best 
known  are  an  aerobic,  liquefying  micrococcus  described  by  Conn,  a 
bacillus  described  by  Weighmann,  Micrococcus  casei  amari  and  Ba- 
cillus liquefaciens  lactis  amari  of  De  Freudenreich  (1895). 

In  fresh  butter  of  good  quality  comparatively  few  microorganisms 
are  found,  but  the  researches  of  Conn  show  that  the  characteristic 
and  agreeable  flavor  of  fresh  butter  is  due  to,  or  at  least  may  be  imi- 
tated by,  a  bacillus  which  is  concerned  in  the  ripening  of  cream 
under  normal  conditions.  Cultures  of  this  bacillus  (Bacillus  41  of 
Conn)  have  already  been  used  in  a  practical  way  by  butter  makers 
to  improve  the  flavor  of  their  product. 

Krenger  (1890)  obtained  from  "cheesy  butter,"  having  a  disa- 
greeable odor,  various  bacteria.  Among  these  the  most  numer- 
ous were  an  oval  micrococcus  (Micrococcus  acidi  lactici,  Kreuger),  a 
slender  bacillus  resembling  Bacillus  fluorescens,  and  Bacillus  acidi 
lactici  of  Hueppe. 

Klecki  (1894)  has  isolated  from  rancid  butter  several  bacteria  not 
previously  described,  one  or  more  of  which  are  no  doubt  concerned 
in  the  production  of  the  rancid  taste  and  odor.  These  are  described 
under  the  following  names:  Bacillus  butyri,  Diplococcus  butyri,  a 
bacillus  resembling  lodococcus  vaginatus  of  Miller,  Tetracoccus 
butyri,  Bacillus  butyri  No.  2. 

Duclaux  (1887)  has  isolated  from  different  kinds  of  cheese  no  less 
than  eleven  different  species  of  bacteria,  which  he  believes  are  con- 
cerned in  the  "ripening  process."  Seven  of  these  are  aerobic  and 
four  anaerobic  species.  Adametz  (1889)  has  also  isolated  and  studied 
a  number  of  species  to  which  he  attributes  the  ripening  of  cheese. 

More  recently  Henrici  (1895)  has  studied  the  bacterial  flora  of 
cheese,  and  Marchal  (1895)  has  shown  that  the  ripening  of  certain 
kinds  of  cheese  (fromages  mous)  is  probably  due  to  Oidinm  lactis. 

Meats,  even  when  salted  and  smoked,  may  contain  living  patho- 
genic bacteria  which  were  present  prior  to  the  death  of  the  animal, 
and,  when  not  properly  preserved,  are  of  course  liable  to  be  invaded 
by  putrefactive  bacteria. 


BACTERIA   IN   ARTICLES   OF  FOOD.  671 

The  researches  of  Foster  (1889)  show  that  the  typhoid  bacillus, 
the  pus  cocci,  the  tubercle  bacillus,  and  the  bacillus  of  swine  plague 
resist  the  action  of  a  saturated  solution  of  salt  for  weeks  and  even  for 
months;  and  the  same  observer  found  that  the  ordinary  processes  of 
salting  and  smoking  did  not  destroy  the  tubercle  bacillus  in  the  flesh 
of  a  cow  which  had  succumbed  to  tuberculosis.  Beu  has  made  cul- 
tures from  a  large  number  of  specimens  of  fresh,  salted,  and  smoked 
meats  and  fish,  with  the  general  result  that  the  fresh  and  salted  meats 
were  found  to  contain  a  limited  number  of  bacteria  of  various  species, 
and  that  smoking  for  several  days  did  not  insure  the  destruction  of 
these  microorganisms.  In  specimens  of  sausage  six  days'  smoking 
did  not  destroy  a  liquefying  bacillus  which  was  present,  but  at  the 
end  of  six  weeks'  exposure  to  smoke  this  bacillus  no  longer  grew, 
while  a  non-liquefying  bacillus  present  in  the  same  specimen  had  not 
been  destroyed.  Fourteen  days'  smoking  sufficed  to  destroy  all  the 
microorganisms  in  a  specimen  of  bacon,  but  this  was  not  sufficient 
for  the  interior  portions  of  a  ham.  Among  the  bacteria  obtained  by 
Beu  from  smoked  meats  he  mentions  the  following  :  Staphylococcus 
cereus  albus,  Proteus  vulgaris,  Staphylococcus  pyogenes  aureus,  Ba- 
cillus liquefaciens  viridis,  etc.  The  number  of  colonies  which  de- 
veloped from  a  fragment,  the  size  of  a  mustard  seed  to  that  of  a  flax- 
seed,  taken  from  the  interior  of  the  meats  examined,  was  usually 
small;  and  the  presence  of  a  few  scattered  bacteria  of  these  common 
species  has  no  significance  from  a  sanitary  point  of  view,  except  as 
showing  that  pathogenic  bacteria  may  survive  in  infected  meats  after 
they  have  been  exposed  to  the  usual  processes  of  salting  and  smoking. 

Petri,  in  experiments  upon  the  bacillus  of  swine  plague  (Schweine- 
rothlauf),  arrived  at  the  following  results  : 

The  flesh  of  swine  which  died  of  this  disease  preserved  its  infec- 
tious properties  after  having  been  preserved  in  brine  for  several 
months,  and  the  same  flesh  salted  or  pickled  for  a  month  and  then 
smoked  for  fourteen  days  contained  the  rothlauf  bacillus  in  a  living 
and  unattenuated  condition.  At  the  end  of  three  months  virulent 
rothlauf  bacilli  were  still  obtained  from  a  smoked  ham,  but  they  were 
no  longer  found  at  the  end  of  six  months. 

Schrank  (1888)  has  made  cultures  from  both  the  albumin  and  the 
yolk  of  fresh  eggs,  and  finds  that  they  are  free  from  bacteria.  He 
thinks  that,  as  a  rule,  putrefactive  bacteria  obtain  access  to  the  inte- 
rior through  injured  places  in  the  sheh1,  although  exceptionally  the 
egg  may  be  infected  with  them  in  the  oviduct  of  the  fowl.  The  usual 
bacteria  concerned  in  the  putrefactive  changes  in  eggs  are,  according 
to  the  author  mentioned,  a  variety  of  Proteus  vulgaris  and  Bacillus 
fluorescens  putidus. 

Zorkendorfer  (1893)  has  cultivated  from  rotten  eggs  sixteen  dif- 


072  BACTERIA   IN   ARTICLES   OF   FOOD. 

ferent  bacilli,  all  of  which  are  described  in  detail  and  none  of  which 
were  found  to  correspond  with  previously  described  species  as  given 
in  Eisenberg's  Bacteriological  Diagnosis. 

Peters  (1889)  has  studied  the  flora  of  the  "sauerteig"  used  in 
Germany  as  yeast  for  leavening  bread.  In  addition  to  the  numerous 
cells  of  three  species  of  Saccharomyces,  he  finds  that  bacilli  are  present 
in  great  numbers,  as  shown  by  direct  microscopical  examination  and 
culture  experiments.  He  describes  five  species,  designated  Bacillus 
A,  B,  C,  D,  and  E,  which  are  commonly  present,  and  to  which  the 
acid  fermentation  of  the  dough  is  ascribed. 

In  Graham  bread  which  had  undergone  changes  making  it  unfit 
to  eat,  Kratschmer  and  Niemilowicz  have  found  the  Bacillus  mes- 
entericus  vulgatus,  which  appears  to  have  been  the  cause  of  the 
fermentation,  which  was  produced  in  bread  having  a  slightly  alka- 
line reaction  by  inoculating  it  with  a  pure  culture  of  this  bacillus. 
The  infected  bread  has  a  brownish  color,  a  peculiar  odor,  and  be- 
comes sticky  and  viscid. 

Uffelmann  (1890)  has  also  studied  the  bacteria  in  spoiled  rye 
bread,  and  obtained,  in  addition  to  common  mould  fungi,  Bacillus 
mesentericus  vulgatus  and  Bacillus  liodermus. 

Waldo  (1894)  has  shown  that  baking  does  not  sterilize  bread. 
This  was  to  have  been  expected  in  the  case  of  the  spores  of  bacilli, 
but  it  is  somewhat  surprising  to  find  that  two  species  of  Sarcina  and 
two  micrococci  survived  the  baking  process.  In  all  Waldo  obtained 
thirteen  species  of  bacteria  from  the  interior  of  sixty-two  loaves 
examined.  Bacillus  subtilis  and  allied  spore-forming  bacilli  were 
most  frequently  found,  and  the  statement  is  made  that  a  loaf  "  from 
a  low-class,  dirty  bakery  will  almost  invariably  contain  more  living 
bacteria  (or  their  spores)  than  .one  from  a  good,  clean  bakery." 

Lehmann  (1894)  under  the  name  Bacillus  levans  has  described  a 
microorganism  which  closely  resembles  Bacillus  coli  communis.  This 
was  obtained  from  sour  dough,  and  was  believed  to  be  the  cause  of 
the  acid  fermentation  which  so  often  interferes  with  success  in  ob- 
taining sweet  and  wholesome  bread.  When  a  culture  of  this  bacil- 
lus was  added  to  flour  and  water,  without  the  addition  of  yeast,  an 
active  fermentation  occurred  and  the  dough  became  acid. 


INDEX 


ABBOTT'S   study  of    pseudo-diphtheritic 

bacilli,  450 

Abel's  capsule  bacillus,  561 
Abriu,  262,  265,  267 
Abscesses,  formation  of,  223 
acute,  micrococci  in,  14 

micrococcus    pneumonise    crou- 

posae  in,  401 

Absinthe,  antiseptic  power  of,  203 
Acetic  acid,  action  of,  on  spores,  182 

as  a  decolorizer  in  staining,  28 
Aceton-celluloidm  solution,  36 
Acetone,  gerniicidal  power  of,  197 
Acetylene  gas  in  photomicrography,  110 
Acid  media,  bacterial  growth  in,  166 
Acids,  action  of,  on  bacteria,  180 
produced  by  bacteria,  137 
in  culture  media,  46 
prejudicial    to   the   vitality   of  the 

cholera  spirillum,  600 
diluted,  as  decolorizers  in  staining, 

28 

Acne  contagiosa  of  horses,  bacillus  of,  557 
Aerobic  bacteria,  16,  172 

in  stab  cultures.  70 
Agar  as  a  medium  of  growth  for  : 
bacillus  A  of  Booker,  555 

aerogenes  capsulatus,  589 

of  Babes  and  Oprescu,  526 

Beck,  566 

of  Belfonti  and  Pascarola,  508 

capsulatus,  522 

capsule,  of  Nicolaier,  560 

of  Cazal  and  Vaillard,  525 

of  cholera  in  ducks,  503 

dysenterise,  569 

enteritidis,  520 

of  Fiocca,  548 

gallinarum,  521 

of  grouse  disease,  521 

43 


Agar  as  a  medium  of  growth  for: 
bacillus  of  hog  cholera.  504,  509 
of  Laser,  524 
of  Lucet,  526 
piscicidus,  567 
piscicidus  agilis,  565 
of  purpura  haemorrhagica,  Kolb, 

559 
of  purpura  hsemorrhagica,  Tiz- 

zoni  and  Giovannini,  558 
pyocyaneus  ,3,  545 
pyocyaneus  pericarditidis,  546 
cedenmtis  maligni.  No.  II.,  588 
solanacearum,  572 
of  symptomatic  anthrax,  586,  587 
of  swine  plague,  Marseilles,  509 
typhi  muriuni,  524 
micrococcus  tetragenus,  412 
proteus  of  Karlinski,  552 
spirillum  of  Finkler  and  Prior,  603 
Metschnikovi,  605 
tyrogenum,  604 

Agar-agar  as  a  solid  culture  medium,  43 
characters  of  growth  in  stab  cultures 

in,  72 
bacillus  of  Emmerich  and  Weibel  in, 

566 

bacillus  pestis  in,  563 
Agar-ge latin,  44 

growth  of  bacillus  pestis  in,  563 
bacillus  acidiformans,- 588 
Agar,  nutrient,  Abbott's  method  of  pre- 
paring, 46 

in  analysis  of  water,  631 
as  a  culture  medium  for : 

bacillus  of  acne   contagiosa  of 

horses,  557 
bacillus  alvei,  557 
bacillus  coli  communis,  531 
bacillus  diphtherias,  454 


674 


INDEX. 


Agar  as  a  culture  medium  for : 

bacillus  endocarditidis  griseus, 

556 

bacillus  erysipelatos  suis,  513 
bacillus  gracilis  cadaveris,  560 
bacillus  leporis  lethalis,  541 
bacillus  cedeniatis  uialigni,  582 
bacillus  pyocyaneus,  543 
bacillus  typhi  abdominalis,  439 
micrococcus  botryogenus,  413 
micrococcus  melitensis,  421 
proteus  horninis  capsulatus,  519 
proteus  lethalis,  555 
proteus  septicus,  554 
proteus  vulgar  is,  549 
nutrient,  Shultz's  method  of  prepar- 
ing, 45 
spirillum   choleras  asiaticae   in, 

596 
staphylococcus  pyogenes  aureus 

in,  374 

streptococcus  pyogenes  in,  384 
plates,  bacillus  anthracis  on,  425 

bacillus  endocarditidis  capsula- 
tus on,  556 
bacillus  cedematis   nialigni  on, 

583 
micrococcus  pneumonias  croupo- 

sae,  403 

solutions,  filtration  of,  44 
stick  cultures,  bacillus  of  purpura 

haemorrhagica,  Babes,  558 
Age,  as  influencing  susceptibility  to  dis- 
ease, 233 
Agitation,    influence    of,    on     bacterial 

growth,  163 

Agua  coco  as  a  culture  medium,  39 
analysis  of,  40 
bacillus     cavicida    havaniensis 

in,  517 

bacillus  cuniculicida  havanien- 
sis in,  538 
Air,  demonstrations  of  micro-organisms 

in,  615 

list  of  saprophytes  found  in,  624 
optically  pure,  5 
of  city  streets,  bacteria  in,  625 
of  closed  schoolrooms,  bacteriology 

of,  625 
of  hospitals,  pathogenic  germs  in, 624 


Albuminous  fluids,  germicidal  powers  of, 

210 

Alcohol  as  a  decolorizer  in  staining,  28 
action  of,  on  bacterial  growth,  197 
on  micrococcus  pneumonias  crou- 

posae,  405 

absolute,  action  of,  on  bacillus  tuber- 
culosis, 478 
Alessi's  experiments  on  the  pathogenic 

action  of  typhoid  bacilli,  447 
Alexins  (Buchner),  236,  265 
Algerian   sheep,    insusceptibility   of,  to 

anthrax,  2o4 

Alkalies,  action  of,  on  bacteria,  180 
Alkalinity  of  blood  as  affecting  immu- 
nity, 236 

Alkaloid  in  anthrax  cultures,  428 
Almonds,    bitter,    antiseptic    power   of, 

203 

Alpha-naphthol,  antiseptic  power  of,  206 
Alum,  antiseptic  value  of,  188 
Aluminum  acetate,  antiseptic  value  of, 

188 

chloride,  antiseptic  value  of,  188 
Amann's  method  of  separating  tubercle 

bacilli  from  sputum,  486 
Ammonia,  action  of,  on  bacterial  growth, 

184 

as  a  nutrient  of  bacteria,  126 
oxidation  of,  by  bacteria,  144 
Ammonium  carbonate,  antiseptic  value 

of,  188 

fluosilicate,  antiseptic  value  of,  188 
sulphate,  antiseptic  value  of,  188 
Amplification  of  photomicrographs,  106 
Anaerobic  bacteria,  16 

cultivation  of,  80 
in  stab  cultures,  71 
isolation  of,  83 
concerned  in  putrefaction,  143 
Anaesthetics  in  experiments  on  animals, 

99 

Analysis,  chemical,  of  Friedlander's  ba- 
cillus, 124 
of     putrefactive      bacteria    by 

Nencki,  124 

of  purified'tuberculin,  362 
Angelica,  antiseptic  power  of,  203 
Aniline  blue  solution,  Loffler's,  29 
colors  as  stains,  25 


INDEX. 


675 


Ani Hue  colors  for  staining  the  anthrax 

bacillus,  424 
in  culture  media,  46,  64 
dyes,  germicidal  power  of,  197 
Aniliue-geutian-violet     solution,     Ehr- 

lich's,  29 
Aniline-uiethyl-violet  solution,  Ehrlich- 

Weigert,  29 
Aniline  oil,  gerinicidal  power  of,  198 

water  for  staining  solutions,  29 
Animals,  experiments  upon,  96 

inoculations  into  susceptible,  169 
post-mortem  examinations  of  inocu- 
lated, 101 

Anise,  antiseptic  power  of,  203 
Annales  de  1'Insthut  Pasteur,  a  reference 

to,  8 
de    Micrographie,    a    reference    to, 

9 
Anthrax,    causation    of,    discovered    by 

Davaine,  6 

animals  immune  to,  233,  234 
antidotal  effect  of  bacillus  pyocya- 

neus  in,  544 

susceptibility  of  herbivora  to,  234 
bacillus,  422 

general  infection  by,  428 

pathogenesis  of,  428 

specific  toxin  of,  430 

sporeless  varieties  of,  4213 

staining  of,  424 

thermal  death-point  of,  157 

vitality   of,  in  various  waters, 

637 

cultures,  attenuation  of,  277 
chemical  products  in,  428 
toxalbumin  of,  150 
protein.  124 

spores,  formation  of,  424,  426 
influence  of  chlorine  on,  177 
influence  of  sunlight  on,  161 
survival   of,   in  various  media, 

430 
symptomatic,  347 

bacillus  of,  585 
Antiabrin,  263 
Anticholeraic   inoculations,    Haffkine's, 

302 

harmlessness  of,  305 
Antipneumatoxin,  261,  341 


Antiseptic   agents,  attenuation  of   viru- 
lence by,  131 
power,  164 

determination  of,  165 
values,  Miquel's  tables  of,  186 
Antiseptics,  definition  of,  164 
Antitetanic     blood    serum^    therapeutic 

value  of,  356 
Antitoxic    values,  Brieger   and    Colin 's 

method  of  determining,  358 
Antitoxin,  diphtheria,  314 
from  goat's  in  ilk,  358 
tetanus,  Qurative  power  of,  359 
typhoid,  present  in  the  blood  serum 
of  persons  who  have  recently  suf- 
fered from  the  fever,  367 
Antitoxins,  237 

bactericidal  value  of,  267 
discovery  of,  259 
immunity  dependent  on,  267 
Apparatus,  sterilization  of,  53 

for  coagulating  blood  serum,  58 
for  filtering  nutrient  agar-agar,  4^ 
for  hydrogen   in    cultivating    anae- 
robic bacteria,  86 
incubation,  of  d' Arson val.  94 
for  photomicrography,  106 
for  sterilizing  by  filtration,  59,  60 
Aqueous  humor,  germicidal  power  of,  236 
Arloing's  experiments  on  spore   forma- 
tion, 160 
Aromatic     products    of    decomposition, 

germicidal  power  of,  198 
Aronson's  diphtheria  antitoxin,  314 
Arsenious  acid,  action  of,  on  bacteria, 

183 

Arthritis  of  the  wrist,  inicrococcus  pneu- 
moniae  crouposse  in.  402 
Arthrobacterium  of  Hueppe,  123 
Arthrospores,  16,  19,  123 

of  the  comma  bacillus,  554,  598 
Artificial  culture  media,  40 
Asbestos,  filtration  of  air  through,  619 
Ascitic  fluid,  germicidal  power  of,  236 
Ascococcus,  general  characters,  17 
grouping  of,  21 
Johnei  (Cohn),  412 
Aseptol,  germicidal  power  of,  198 
Atmosphere,    the,   relatively   free    from 
bacteria,  613,  622 


C76 


INDEX. 


Attenuated  virus,  Pasteur's  method  of 

obtaining,  244 

restoration  of  virulence  to,  245 
of  anthrax,  427 

cultures  of  the  anthrax  bacillus,  429 
Attenuation  of  virulence,  130 

by  cultivation    in   the  blood   of  an 

immune  animal,  132 
of  the  bacillus  of  diphtheria,   457, 

458 

of  the  bacillus  of  tuberculosis.  3(?0 
of  cultures  of  bacillus  mallei,  4i>4 
of  the  microbe  of  fowl  cholera,  502 

JJABES,  on  the  pigments  of  bacillus  pyo- 

cyaneus,  134 
Babes'  bacillus  of  purpura  hsemorrhagica, 

558 

Bacille  du  charbon  symptomatiqne,  585 
de  la  morve  (B.  mallei),  489 
du  rouget  du  pore  (Pasteur),  511 
virgule  cholerigene,  593 
Bacillen  des  griinblauen  Eiters,  542 
Bacilli,  general  characters,  18 
morphology,  22 
anaerobic  (pathogenic),  578 
in  chronic  infectious  diseases,  467 
movements,  120 
of  Baumgarten,  12 
non-pathogenic,  list  of  species  found 

in  the  soil  (Fillies),  646 
list  of  species  found  in  water, 

638 
pathogenic  (anaerobic),  578 

list  of  species   found    in   soils 

(Fttlles),  646 
list  of  species  found  in  water, 

639 

pseudo-typhoid,  441 
which  produce  septicaemia  in  sus- 
ceptible animals,  498 
Bacillus  A  of  Booker  (from  cholera  in- 

fantuni),  555 

aceticus,  acid  produced  by,  137 
acidiformans  (Sternberg),  139/537 

pathogenesis  of,  538 
acidi  lactici,  667 

thermal  death-point  of,  155 
of  acne  contagiosa  of  horses,  557 
aerogenes  capsulatus  (Welch),  587 


Bacillus  alvei,  556 

pathogenesis,  557 
arnylovorus  (Burrill),  575 
anthracis,  422 

biological  characters,  424 
destroyed  by  gastric  juice,  658 
morphology  of,  423 
pathogenesis,  428 
spore  formation,  426 
resistance  of,  to  high  tempera- 
tures, 154 

thermal  death-point  of,  150 
vitality  of,  in  waters  of  various 

characters,  637 
vitality  of,  when  buried  in  the 

soil,  645 

of  anthrax,  symptomatic,  585 
biological  characters,  586 
methods  by  which  virulence  is 

increased,  587 
morphology,  586 
pathogenesis  of,  587 
vital  resistance  of  its  spores,  586 
B  of  (Booker),  536 
of  Babes  and  Oprescu,  525 
of  Beck,  from  infected  rabbits,  566 
of  Belfanti  and  Pascarola,  508 
der  Brustseuche    beim    Kaninchen, 

566 
der  Bliffelseuche   (Oreste-Armanni), 

499 

butyricus,  acid  produced  by,  138 
cadaveris  (Sternberg)    from  yellow- 
fever  cadavers,  584 
grandis  (Sternberg)  of  putrefac- 
tion, 665 

campestris  (Pammel),  575 
capsulatus,  521 

inucosus  (Fasching),  561 
cavicida,  516 

liavaniensis  (Sternberg),  516 
thermal  death-point  of,  155 
vitality     of,     in    moist    atmos- 
pheres, 159 

of  Cazal  and  Vaillard,  525 
of  cholera  (see  Spirillum),  593 
of    cholera    in    ducks    (Cornil    and 

Toupet),  503 

cholerae  gallinarum  (Fliigge),  499 
coli  communis,  529 


INDEX. 


G77 


Bacillus  coli  commimis,  probably  identi- 
cal with  bacillus  cavicida,  529 
commimis,  discovery  of,  530 
commimis,     biology    and    mor- 
phology of,  530 

commimis,  pathogenesis  of,  532 
communis.  varieties  of,  533 
coprogenes  parvus.  515 
crassus  sputigenus.  517 

sputi genus,  thermal  death-point 

of,  155 
cuniculicida  (Fliigge),  499 

havaniensis  (Sternberg),  538 
havanieusis  pathogenesis,  540 
cyanogenus.  thermal  death-point  of, 

155 
diphtheria,  449.  453 

biological    and     morphological 

characters,  4~>'-\ 
co  1  u  in  bar  u  in,  458 
columbarum,  pathogenesis,  455 
columbarum,      thermal      death- 
point  of,  455 
vitulorum.  4-V.i 
dysenteric  (Shiga),  568 

distinguished  from  bacillus  ty- 

phosus,  569 
of  Eberth,  431 

of  Emmerich  and  Weibel  from  in- 
fected trout,  565 
endocarditidis  capsulatus,  550 

griseus,  555 

enteritidis  (Gartner),  520 
erysipelatos  suis,  511 

suis,  biological  characters,  512 
suis,  morphology,  511 
suis,  pathogenesis,  514 
suis,  action  of  germicides  on,  513 
of  Fiocca,  from  saliva  of  cats  and 

dogs.  547 
fluorescens,  thermal  death-point  of, 

155 
pntridus,  influence  of  light  on, 

161 

of  foul  brood  (of  bees),  556 
of  fowl  cholera,  499 

thermal  death-point  of,  501 
action  of  antiseptics  on.  501 
der     Frettchenseuche     (Eberth    and 
Schimmelbusch).  o08 


Bacillus  of  Friedlander.  390 

gallinarum  (Klein) not  identical  with 
Pasteur's  bacillus  of  fowl  chol- 
era, 521 

thermal  death-point  of,  150 
gracilis    cadaveris  (Sternberg)  from 

liver  of  man,  560 
of  green  pus,  542 
of  grouse  disease  (Klein),  520 
of  hog  cholera  (Salmon  and  Smith), 

503 

pathogenesis,  505 
der  Hlilinercholera.  499 
hyacinthi  (Walker),  573 
hydrophilus  fuse  us  (Sanarelli),  522 
indicus.  thermal  death-point  of,  155 
of  influenza  (Pfeiffer),  403 

toxin  of,  405 

der  Kanincheuseptikamie(Koch),  499 
lactis  aerogenes  (Escherich),  536 
of  Laser,  perhaps  identical  with  the 
bacillus    of    swine    plague,   Mar- 
seilles, 524 
leporis  lethalis  (Sternberg,  Gibier), 

541 
leprae,  486 

pathogenesis,  488 

of  Lucet,  perhaps  identical  with  ba- 
cillus gallinarum  of  Klein,  526 
mallei,  489 

pathogeuesis,  491 
thermal  death-point  of,  156 
des  Mauseseptikamie  (Koch),  511 
of  Mereshkowski,  565 
of  mouse  septicaemia,  511 

action  of  chlorine  on,  177 
mucosus  ozffinae  (Abel),  561 
murisepticus  (Fltigge),  511 

thermal  death-point  of,  155 
pleomorphus  (Karlinski),  552 
neapolitauus,    thermal     death-point 

of,  155 

cedematis  malign i,  580 
maligni,  biology.  582 
malign  i,  morphology,  581 
maligni,  pathogenesis,  583 
maligni,  No.  II.  (Novy).  587 
pestis  (Kitasato  and  Yersin),  562 
biological  characters  as  given  by 
Kitasato.  563 


678 


INDEX. 


Bacillus  pestis,  influence  of  desiccation 

on,  564 
piscicidus  (Fischel  and  Enoch),  567 

agilis  (Sieber),  565 
pneumonias  (Fliigge)   (see  Micrococ- 

cus),  396 

thermal  death-point  of,  155 
of  preputial    sniegnia    similar  mor- 
phologically to  the  syphilis  bacil- 
lus. 405 

prodipio8us,pigment  produced  by,  135 
influence  of  electric  currents  on, 

163 

influence  of  light  on,  161 
influence    on    the  virulence    of 

anthrax  cultures,  587 
thermal  death-point  of,  155 
vitality  of,  in  moist  atmospheres, 

159 
of  purpura  hsemorrhagica  of  Babes, 

558 

hsemorrhagica  of  Kolb,  559 
haemorrhagica  of    Tizzoni    and 

Giovanini,  558 
pyocyaneus,  542 

Mitidotal  effect  of,  in  inoculated 

anthrax,  544 

butyric  acid  produced  by,  139 
green  pigment  of,  134,  543 
general  infection  by,  544 
pathogenesis  of,  543 
thermal  death-point  of,  155 
varieties  of,  545 
pyocyaneus  ft  (P.  Ernst),  545 

distinguished     from     bacillus 

pyocyaneus,  545 
pyocyaneus    pericarditidis    (H.     C. 

Ernst),  545 
pyogenes  liliformis  (Flexner),  567 

fcetidus  (Passet),  518 
of  rabbit  septicaemia,  499 
of  rhinoscleroma,  496 

similarity    of,  to  Friedlander's 

bacillus,  497 
der  Rinderseuche  (Kitt),  499 

amerikanischen  (Caneva),  508 
salivarius  septicus  (Biondi),  398 
septiceemiae  haemoiThagicaB,  499 

hsemorrhagicse.  biology  and  mor- 
phology of,  500 


Bacillus  septicsemise  haemorrhagicse,  va~ 

rieties  of,  344 
septicus  agrigenus  (Nicolaier),  510 

sputigenus  (Fliigge),  398 
des    Schweinerothlauf   (Loffler    and 

Schutz),  511 

thermal  death-point  of,  155 
der     Schweineseuche    (Loffler     and 

Schutz),  499 

Marseilles  (Rietsch-Jobert),  508 
solanacearum  (Smith),  571 
of    spontaneous    rabbit    septicaemia 

(Eberth),  508 

of  swine  pest  (Selander),  503 
plague  (Billings),  503 
plague,  Marseilles,  508 
post-mortem      appearances      in 

death  from,  510 

subtilis,  resistance   of,  to  high  tem- 
perature, 154 

tenuis  sputigenus  (Pansini),  523 
tetani,  578 

tracheiphilus  (Smith1),  576 
tuberculosis,  468 

attenuation  of,  485 
biological  characters,  472 
branching  forms  of,  484 
chlorine,  action  of,  on,  177 
cultures,  difficulties  of  obtaining 

pure,  474 
cultures,  methods  of  obtaining, 

476 
gallinarum,   not  a  variation   of 

bacillus  tuberculosis,  482 
germicides,  action  of,  on,  478 
light,  action  of,  on,  161 
morphology,  469 
in  nasal  cavity,  healthy,  485 
pathogenesis,  480 
reproductive  elements  of,  485 
resistance  of,  to  freezing,  153 
not  ordinarily  saprophytic,  474 
spore   formation  undetermined, 

472 

sputum,  examination  of.  for,  470 
sputum,    estimation  of  number 

of,  in,  479 

staining  methods  for,  30 
thermal  death-point  of,  158 
vitality  of,  478 


INDEX. 


679 


Bacillus,  typhi  abdominalis  (see  Typhoid 
Bacillus),  430 

murium,  524 
typhosus,  431,  436 
ureae,  140 

der  \Vildseuche  (Hueppe),  499 
X  (Steinberg)  from  yellow- fever  ca- 
davers, 538 
Bacteria,    a  distinct  class  of   vegetable 

organisms,  11 
action  of  acids  on,  180 

of  alkalies,  183 

of  coal-tar  products,  197 

of  gases,  172 

of  the  haloid  elements,  172 

of  salts,  186 

chemical  composition  of,  124 
classification  of,  10 
conditions  of  growth  of,  125 
culture  media  for,  37 
dependent  on  pabulum,  153 
dimensions  of,  20 

influence  of  pressure  on  growth  of, 
163 

of  physical  agents,  153 

of  one  species  on  the  growth  of 

another,  128 

mistaken  for  infusoria,  3,  10 
modes  of  grouping,  20 
modifications  of   biological  charac- 
ters, 129 

morphology  of,  20 
motions  of,  117 
the  photographing  of,  103 
probably  seen  by  Leeuwenhoeck,  3, 10 
products  of  vital  activity,  133 

acids,  137. 

butyric  acid,  138 

fermentation,  136 

fermentation,  alkaline  in  urine 
141 

phosphorescence,  145 
a  possible  nucleus  in,  117 
reproduction  of,  117 
rapidity  of  reproduction,  120 
structure  of.  117 
thermal  death-point  of,  155 
varieties  of,  117 

vitality   of,    in  moist  atmospheres, 
159 


Bacteria  in  the  air,  G13 

number   of   relatively  unimpor- 
tant, 624 

in  articles  of  food,  667 
in  croupous  pneumonia,  396 
in  diphtheria,  449 
in  the  dust  of  city  streets,  646 
in  the  soil,  methods  of  studying,  642 
action  of,  on  buried  pathogenic 

bacteria,  645 
number  of,  in  various  localities 

and  at  various  depths,  643 
of  graveyards,  643,  646 
in  water,  62*  > 

species  very  numerous,  636 
distilled,  125 

of  cadavers   and  of  putrefying  ma- 
terial from  various  sources,  664 
of  plant  diseases,  571 
of    the    skin    and  exposed    mucous 

membranes,  648 
species  found  on,  649 
of  the  stomach  and  intestine,  658 
nitrifying,  144 
non-pathogenic,  thermal  death-point 

of,  157 
parasitic,  127 
pathogenic,  fatal  dose  of,  242 

modes  of  action  of,  219 
Bacteriaceae  of  Zopf,  12 
Bacterial  diseases,  immunity  afforded  by 

an  attack,  243 
Bacte'ridie  du  charbon,  423 
Bacteriology,  literature  of,  8 

early  progress  of,  6 
Bacterium  aeruginosum,  542 

coli  commune  (Escherich),  516,  529 
of  Davaine's  septicaemia,  499,  500 
of  Ehrenberg,  3,  10 
termo,  548 

Barium  chloride,  antiseptic  value  of,  188 
Baurngarten,  bacilli  of,  12 

classification  of  bacteria,  12 
on  the  influence  of  Koch's  tubercu- 
lin, 364 

Bedding,  disinfectants  for,  213 
Beef  extracts  as  culture  media,  41 
Beggiatoa,  sulphur  in,  24 
Behring's  discovery  of  the  antitoxins.  8 
antitoxin  of  diphtheria,  314 


680 


INDEX. 


Behring's  discovery  of  the  immunization 

of  large  animals  against  tetanus, 356 

serum  for  tetanus,  strength  and  doses 

of,  357 
standard  of  diphtheritic  antitoxin, 

313 
Belfonti's   experiments   on   micrococcus 

pneumonia;  crouposse,  341 
Benzene,  germicidal  power  of,  198 
Benzoic  acid,  antiseptic  value  of,  183 
Benzo-naphthol,    no    germicidal    power, 

206 

Beta-naphthol,  antiseptic -power  of,  206 
Binary  division  of  bacteria,  120 
Biological  classification  of  bacteria,  13 
Biskra  button,  micrococcus  of,  415 
Bismarck  brown  as  a  stain,  25 

for  staining  anthrax  bacilli,  424 
Blackleg,  347,  585 
Bladder,  urinary,  free  from  bacteria  in 

health,  654 

Blood,  alkalinity  of,  as  affecting  immu- 
nity, 236 

free  from  bacteria  in  health.  664 
germicidal  power  of,  236 
of  typhoid  patients  does  not  yield 

cultures,  434 
serum  as  a  culture  medium,  -'57 

germicidal  powers  of,  208,  257 
immunizing  value  of,  313 
mixture,  Loffler 's,  48 
solidification  of,  57 
solidified,  streak  cultures  on,  77 
sterilization  of,  57 
Sternberg's    method  of  collect- 
ing, 38 

Blood  serum  as  a  medium  of  growth  for . 
bacillus  A  of  Booker,  555 
bacillus  of  acne  contagiosa  of 

horses,  557 

bacillus  anthracis,  426 
bacillus  coli  communis,  531 
bacillus  endocarditidis  griseus, 

556 

bacillus  enteritidis,  520 
bacillus  erysipelatos  suis,  513 
bacillus  of  Friedlander,  397 
bacillus  hydrophilus  fuscus,  523 
bacillus  mallei,  491 
bacillus  cedematis  maligni,  582 


Blood  serum  as  a  medium  of  growth  for: 
bacillus  pestis,  563 
bacillus  of  purpura  hsemorrhag- 

ica  (Baoes),  558 

bacillus  of  purpura  hsemorrhag- 
ica  (Tizzoni  and  Giovannini), 
558 

bacillus  of  swine  plague,  Mar- 
seilles, 509 
bacillus  tetani,  580 
bacillus  tuberculosis,  474,  475 
bacillus  typhi  abdominalis,  439 
micrococcus  gonorrhoeas,  393 
micrococcus  Manfredi,  413 
proteus  hominis  capsulatus,  519 
proteus  of  Karlinski,  552 
proteus  lethalis,  555 
proteus  septicus,  554 
spirillum  cholerse  asiaticse,  596 
spirillum  of  Finkler  and  Prior, 

603 
Bluchers  apparatus  for  an  atmosphere  of 

hydrogen,  87 
Blue  milk,  668 

rays  of  spectrum,  germicidal  power 

of,  161 

Bonn's  thernio-regulator,  90 
Boiling  in  water,  as  a  disinfectant,  212, 

213 
Booker's  bacillus  B,  probably  identical 

with  bacillus  lactis  aerogenes,  536 
Boracic  acid,  action  of,  on  bacteria,  182 
Borden's  method  of  photographing  by  oil 

light,  111 
Bordoni-Uffreduzzi  on  the  bacteria  of  the 

human  skin,  648 
bacillus  of,  from  the  marrow  of 
the  bones  of  a  leper,  probably 
not    identical    with    bacillus 
leprse,  487 
Boric   acid,    action   of,   on   micrococcus 

pneumonise  crouposae,  405 
Botkin's  apparatus  for  an  atmosphere  of 

hydrogen,  87 
bacillus  butyricus,  139 
Bougies  of  the  Chamberland  filter,   59, 

60 
Bouillon  as  a  culture  medium,  41 

for  bacillus  aerogenes  capsula- 
tus. 589 


INDEX. 


681 


Bouillon  as  a  culture  medium  for  bacil- 
lus of  Babes  aud  Oprescu,  526 
for  bacillus  of  Beck,  566 
for  bacillus  of  Cazal  and  Vail- 

lard,  525 
for     bacillus     coli     communis, 

532 

for  bacillus  diphtherias,  454 
for  bacillus  dysenteriae,  569 
for  bacillus  of  Emmerich  and 

Weibel,  566 
for    bacillus   erysipelatos  suis, 

513 

for  bacillus  of  Fiocca,  548 
for  bacillus  galliuarum,  521 
for   bacillus  gracilis  cadaveris, 

560 
for  bacillus  of  hog  cholera,  504, 

509 

for  bacillus  of  influenza,  464 
for  bacillus  of  Laser,  524 
for  bacillus  of  Lucet,  526 
for   bacillus   of  Mereshkowsky, 

565 

for  bacillus  of  Nicolaier  (cap- 
sule), 560 
for  bacillus  cedematis  maligni, 

No.  II.,  588 

for  bacillus  piscicidus,  567 
for  bacillus  pyocyaneus  pericar- 

ditidis,  546 
for  bacillus   septicaemia   haem- 

orrhagicae,  501,  509 
for  bacillus  solanacearum,  572 
for    bacillus    of    swine  plague, 

Marseilles,  509 
for  bacillus  tetani,  579 
for    bacillus    of    von    Dungern 

(capsule),  562 

for  micrococcus  Manfredi,  413 
for  proteus  hominis  capsulatus, 

519 
for  spirillum  cholerae  asiaticee, 

596 
for  spirillum  of  Metschnikovi, 

605 
Bovine  mastitis,  micrococcus  of  (Kitt), 

414 

pneumonia,  micrococcus  of,  414 
Branching  forms  of  bacilli,  123 


Bread,  not  sterilized  by  the  heat  of  bak- 
ing, 672 

paste  as  a  culture  medium,  49 
Brieger's  bacillus,  516 

toxic  ptomains  from  cholera  cultures, 

21)2 

typhotoxin,  367 
Bromine,     influence     of,    on     bacterial 

growth,  178 
Bronchial  glands,  living  tubercle  bacilli 

found  in,  485 
Broncho-pneumonia,   a  pseudo-influenza 

bacillus  in,  466 
Brownian  movements,  119 
Brustseuche,  335 
Bubonic  plague,  562 

discovery  of  the  bacillus  of,  8 
infection  in  man  by  inoculation 
through  cutaneous  lesions  and 
by   the   respiratory   passages, 
565 
rats  and  fleas  as  propagators  of, 

564 

Buchner's  method  of  cultivating  anaero- 
bic bacteria,  85 
theory  of  immunity,  255 
Bujwid's  test   for  the  presence   of  the 

cholera  spirillum,  598 
experience  with  tuberculin,  365 
method  of  preparing  tuberculin,  363 
Bulbs,  Steruberg's,  38,  66 
Bureau   of   Animal   Industry,   report  of 
measures  for  arresting  swine  plague, 
345 

Butter,  bacteria  in,  670 
Butyric  acid,  action  of,  on  bacteria,  183 
produced 'by  bacteria,  138 

CADAVERIN,  146 

Cajuput,  antiseptic  power  £»f,  203 
Calamus,  antiseptic  power  of,  203 
Calcium  chloride,  antiseptic  value  of,  188 
hydroxide,    action   of,    on  bacterial 

growth,  184 
hypochlorite,    antiseptic    value    of, 

188 

light  in  photomicrography,  107 
Camphor,  germicidal  power  of,  199,  203 
Canada   balsam   for  mounting  bacterial 
slides,  28 


682 


INDEX. 


Canon's  method  of  demonstrating  the  ba- 
cillus of  influenza,  463 
Capsule  bacilli,  Nicolaier's  comparison 

of  the  characters  of,  560 
bacillus  of  Nicolaier,  560 
of  von  Dungern,  562 
Caraway,  antiseptic  value  of,  203 
Carbolic  acid  in  culture  media,  47 
as  a  disinfectant,  212,  213 
action    of.    on    anthrax   spores, 

587 
action  on  bacillus  tuberculosis, 

478 
action    on     micrococcus    pneu- 

monice  crouposse,  405 
for  restraining  growth  of  water 
bacteria,  in  searching  for  the 
typhoid  bacillus,  444 
Carbol-fuchsin  solution,  Ziehl's,  29 
Carbon  for  bacterial  growth,  126 

dioxide,   influence   of,    on  bacterial 

growth,  174 

prevents  growth  of  the  anaerobic 
bacillus  of  symptomatic  an- 
thrax, 586 

does  not  prevent  growth  of  the 
anaerobic    bacillus  cedernatis 
maligni  No.  II.,  588 
monoxide,  influence  of,  on  bacterial 

growth,  174 

Carnivora  immune  to  tuberculosis,  233 
Carrot,  antiseptic  power  of,  203 
Cars,  railway,  disinfection  of,  214 
Cascarilla,  antiseptic  power  of,  203 
Cattle,  prevalence  of  tuberculosis  among, 

482 

Caucasian  milk  ferment,  139 
Caustic  potash,  action  of,  on  micrococcus 

pneimionise  crouposse,  405 
Cedar,  antiseptic  power  of,  203 
Celery,  antiseptic  power  of,  203 
Cell-globulin,  germicidal  power  of,  210 
Cells,  vital  activity  of,  133 
Cellular  structure  of  bacteria,  117 
Cellulose,  fermentation  of,  141 
"  Centralblatt  fur  Bakteriologie,"  9 
Cerebrospinal     meningitis,    diplococcus 

of,  410 

Cesspools,  disinfection  of,  217 
Chamomile,  antiseptic  power  of,  203 


Chantemesse  and  Widal's  immunization 
against  the  typhoid  bacillus, 
368 

method  of  searching  for  the 
typhoid  bacillus  in  water  sup- 
plies, 444 

Chaplets,  grouping  of  bacteria  in,  22 
Charbon,  bacillus  of,  42U 
symptomatique,  347 

Chauveau's  retention   theory   of   immu- 
nity, 250 
Cheese,  bacteria  in,  670 

toxic  principle  in,  148 
Chemical  agents,  influence  of,  on  cultures 

of  the  cholera  spirillum,  597 
analysis*  of  water,  advantages  of,  631 
Chemiotaxis,  247,  257 
Cherry  laurel,  antiseptic  power  of,  203 
Cheyne's  experiments  on  the  toxicity  of 

cultures  of  proteus  vulgaris,  550 
observations  on  the  pathogenic  power 

of  saprophytes,  529 

Chloral  hydrate,  antiseptic  value  of,  189 
Chloride  of  lime  as  a  disinfectant,  212,  213 
Chlorinated  soda  as  a  disinfectant,  213 
Chlorine,    influence      of,    on    bacterial 

growth,  177 

Chloroform,  action  of,  on  bacteria,  177 
Cholera,  a  disease  of  man,  233 
cultures,  toxalbumin  of,  150 
experimental  evidence  of  its  produc- 
tion in  man  by  the  comma  bacil- 
lus, 601 

immunity  after  an  attack  of,  297 
infection,  heat  as  a  disinfectant  for, 

154 

ptomains,  149 
second  attacks  of,  243 
spirillum,  influence  of  light  on,  162 
vaccine  (Ferran),  299 

(Haffkine),  302 
Cholera  infantum,  bacillus  A  of  Booker 

in,  555 

proteus  vulgaris  in,  550 
Cholera  morbus  not  caused  by  the  Fink- 

ler-Prior  spirillum,  602 
proteus  Hauseri  in,  551 
Cholin,  147 

Chromic  acid,  action  of,  on  putrefactive 
bacteria,  181 


INDEX. 


683 


Chromogenic  organisms,  14,  135 
Cinnamon,  antiseptic  power  of,  203 
Cities,  bacteriology  of  the  air  of,  025 
Citric  acid,  action  of,  or  pathogenic  bac- 
teria, 182 

Citron,  antiseptic  power  of.  203 
Cladothrix,  apparent  branching  in,  24 
Cladotrichese  (Zopf),  general  characters, 

12,  19 

Classification  of  bacteria,  10 
Clostridinm,  general  characters  of,  18 
spore  formation  in,  122 
of  bacilli,  23 

Clothing,  disinfection  of,  213 
Clou  de  Biskra,  micrococcus  of,  415 
Cloves,  antiseptic  power  of,  203 
Cocci  of  Baumgarten,  12 
Coccocese  (Zopf),  12 
Cocoanut  water  analysis  of,  40 
as  a  culture  medium,  39 
for  bacillus  cavicida  havanien- 

sis  in,  517 
for  bacillus  cuniculicida  hava- 

niensis  in,  539 

Coffee  infusion,  antiseptic  power  of,  200 
Cohn's  classification  of  bacteria,  11 

solution,  40,  126 

Cohnheim,  production  of  tuberculosis  by 

inoculation  of  tuberculous  material,  467 

Cold,  influence  of,  on  bacterial  vitality, 

153 
Colon  bacilli,  difficulty  of  distinguishing 

from  allied  groups,  534 
and  typhoid  bacilli,  varieties  of 

the  same  species?  447 
frequency  of,  in  water  supplies, 

640 
methods    for  their  detection  in 

water  supplies,  640 
Smith's  method  for  their  detec- 
tion, 535 
bacillus  of  Escherich,  529 

its    important    role    in    human 

pathology,  533 

Colonies  on  plates  of  solid  media.  72 
Comma  bacillus  of  Koch,  593 
Conjunctiva,  bacterial  flora  of,  649 
Contact  preparations,  27 
Copaiba,  antiseptic  power  of,  203 
Copper  sulphate  as  a  disinfectant,  212 


Coriander,  antiseptic  power  of,  203 
Corn,  Indian,  bacterial  diseases  of,  575 
Cotton,  first  use  of,  as  a  filter  for  micro- 
organisms, 4 
plugs  for  test  tubes,  62 

for  tubes  containing  aerobic  bac- 
teria, 54 

in  growing  tubercle  bacilli,  475 
Cover-glass  preparations,  25 
Cows,  cocci  of  haemoglobinuria  of,  419 

streptococcus  of  mastitis  of,  419 
Creolin,  germicidal  power  of,  201 
Creosote,  germicidal  power  of,  201 
Cresol,  germicidal  power  of,  201 
Cruciferous  plants,  brown  rot  in,  575 
Cuba,  cases  of  Malta  fever  from,  420 
Cucumbers,  bacterial  diseases  of,  576 
Cucurbitacese,  bacterial  diseases  of,  576 
Culture  media,  37 

artificial,  40 
for  bacillus  mallei,  490 
for  bacillus  tuberculosis,  474 
liquid,  40 
natural,  37 
neutralization  of,  49 
solid,  41 

sterilization  of,  52 
Cultures  of  anthrax,  chemical  products 

in,  428 

of  bacillus  diphtherias, vitality  of,  455 
of  typhoid  bacilli  obtained  from  the 
spleen  in  typhoid  fever  cases,  433, 
436 

drop,  64 

in  liquid  media,  62,  67 
in  solid  media,  69 
stab,  69 
streak,  77 

Cumin,  antiseptic  power  of,  203 
Cunningham's   objections    to   accepting 
Koch's    bacillus   as    the    specific 
cause  of  cholera,  601 
vibrios  resembling  those  of  cholera. 

609 

Cupric  chloride,  antiseptic  value  of,  189 
sulphate,  antiseptic  value  of,  189 
action  of,  on  micrococcus  pneu- 

moniae  crouposse,  405 
Curry  on  Malta  fever  in  the  Philippine 
Islands,  420 


684 


INDEX. 


Cystitis,  proteus  vulgaris  in,  551 
Czenzynke's   staining  solution    for   the 
bacillus  of  influenza,  468 

D'ARSOXVAL'S  incubating  apparatus,  04 
Davaine,  causation  of  anthrax  discovered 

by,  0.  422 

classification  of  bacteria,  10 
Dead,  disinfection  of  the.  213 
Death-point,  thermal,  of  bacteria,  155 
De  Bary's  classification  of  bacteria,  12 
Decolorization  in  staining,  28 
Defensive  proteids  of  Haffkine,  265 
De  Giacoma's  method  of  staining  syph- 
ilis bacilli,  495 
Deneke's  spirillum,  603 
De  Schweinitz,  immunity  from  tubercu- 
losis conferred  by  attenuated  cultures, 
364 
Desiccation,    influence   of,   on   bacteria, 

159 

resistance  of  anthrax  spores  to,  427 
influence  of,  on  bacillus  pestis,  564 

on  the  comma  bacillus,  597 
Developers  in  photomicrography,  113 
Diaphtherin,  germicidal  power  of,  202 
Dieudonne  on  the  vibrios  resembling  the 

cholera  spirillum,  606 
Dimethylamine,  147 
Diphtheria  antitoxin,  314 

results  of  treatment  with,  462 
bacillus  of,  found  in  throat  for  weeks 
after  subsidence  of  fever,  451, 
460 

attenuated  varieties,  309 
branching  forms  of  (C.  Frankel), 

460 

bacteria  in,  449 
cultures,  toxalbumin  of,  312 
evidence  on  which  the  Klebs-Lb'ffler 
bacillus  is  regarded  as  the  cause 
of,  450 

heat  as  a  disinfectant  for,  154 
intestinal  in  rabbits,  bacillus  of,  459 
a  local  infection.  440 
mixed  infections  in,  461 
post-mortem  appearances  of,  226 
toxalbumin  of,  140 

Diphtheritic   false   membranes,    strepto- 
coccus pyogenes  in.  386 


Diphtheritic     inflammation,    cause    of, 

225 

Diplococcns,  general  characters,  17 
of  Frankel,  339 
grouping  of,  21 

intracellularis  ineningitidis,  410 
pneumonise,  discovery  of,  7 
pneumonise  (Weichselbaum),  398 
in  meningitis,  400. 
in  otitis  media,  373,  390 
Discontinuous  heating  in  sterilizing,  53, 

56,  57 
Disease,  race  immunity  to,  234 

susceptibility  to,  233 
Diseases  of  plants,  bacteria  of,  571 
Disinfectant,  importance  of  sunlight  as 

a,  102 
Disinfectants,  action  of,  164 

influence  of,  on  cholera  cultures,  507 
Disinfection,  practical  directions  for,  212 
Disinfektol,  germicidal  power  of,  202 
Distilled  water,  bacteria  in,  125,  635 
Double  staining  of  bacterial  preparations, 

28 

of  spore  preparations,  31 
Drop  cultures,  04 

Druse  des  Pferdes.  streptococcus  of,  418 
Ducks,  bacillus  of  cholera  in  (Cornil  and 

Toupet),  503 

Duclaux's  solution  for  butyric-acid  fer- 
ment, 138 

Dunham's  solution,  151 
peptone  solution,  41 
test  for  the  presence  of  the  cholera 

spirillum,  598 

Dust,  atmospheric,  bacterial  constitution 
of,  614 

EBERTH'S  bacillus.  431 
Egg  albumen  as   a  culture  medium   for 
bacillus  pyocyaneuspericarditidis.  547 
Eggs,  bacteriology  of,  671 

as  a  culture  medium,  40 
Ehrenberg's  vibrioniens,  3,  10 
Ehrlich's  aniline-gentian-violet  solution, 

29 
solution  in  staining  bacillus  mallei, 

400 

method  of  staining  bacillus  tubercu- 
losis, 469 


INDEX. 


685 


Ehrlich's  side  chain  theory,  269 
Ehrlich-Weigert's    aniline-methyl-violet 

solution,  29,  471 

method  of  staining  the  tubercle  ba- 
cillus, 30 

stain  for  syphilis  bacilli,  495 
Electric  light  in  photomicrography,  107 
Electricity,    influence    of,    on    bacterial 

growth,  162 

Eisner's   method    for  detecting  the   ty- 
phoid  bacillus   in    water   and    faeces, 
446 
Emmerich's   bacillus  (B.   ueapolitanus), 

529 

thermal  death-point  of,  155 
experiments   on    rnicrococcus   pneu- 
monias crouposae,  341 
Endocarditis,     ulcerative,     micrococcus 

pneumonise  crouposae  in,  401 
Endospores,  16 

Enzymes   produced  by    liquefying    bac- 
teria, 136 
Eosin  as  a  double  stain,  28 

collodion  in  photographing  bacteria, 

103 

Eosinophile  cells,  237 
Epidemics,  Pasteur  on  the  origin  of,  290 
Erlenmeyer  flasks,  60,  63 
Escherich's  view   that  pseudo-diphther- 
itic bacilli  are  distinct  from  the  true 
bacillus,  460 
Esmarch's  roll  tubes,  76. 

tubes    in    water    analysis,  628, 

630 

method  of  cultivating  anaerobic  bac- 
teria, 84 

Essential  oils,  antiseptic  power  of,  202 
Ether,  action  of,  on  bacillus  tuberculosis, 

478 

antiseptic  power  of,  202 
in  experiments  on  animals,  99 
Eugenol,  antiseptic  power  of,  203 
Euphorin,  feeble  germicidal  activity  of, 

204 

Eucalyptol,  antiseptic  power  of,  203 
Eucalyptus,  antiseptic  power  of,  203 
Excreta,  disinfection  of,  212,  217 

method  of  testing  for  the    cholera 

spirillum,  598 
Exhaustion  theory  of  immunity,  249 


Expired  air  free  from  micro-organisms, 

622 
Eye,  inoculations  into,  99 

FACULTATIVE  anaerobics,  16,  18 

in  stab  cultures,  70 
parasites,  15,  127,  221 
saprophytes,  221 

Faeces,  list  of  species  of  pathogenic  bac- 
teria found  in,  663 
Fallopian  tubes  free   from    bacteria  in 

health,  654 

Farcy,  symptoms  of,  491 
Fasching's  capsule  bacillus,  561 
Feet,  the,  bacteria  on,  649 
Fennel,  antiseptic  power  of,  203 
Ferment  lactique  of  Pasteur,  667 
Fermentation  by  bacteria,  136 

of  grape    sugar   in   water    sup- 
plies, 535 

in  liquid  media,  68 
putrefactive,  141 
test  for  colon  and  typhoid  bacilli  in 

water,  445 
tubes,  68 
of  urea,  140 
viscous,  141 
Ferments,  soluble,  produced  by  bacteria, 

143 
Fermi  and  Pernossi  on  the  properties  of 

tetanus  poison,  354 

Ferran's  anticholeraic  inoculations,  301 
Ferret  disease,  bacillus  of,  508 
Ferric    chloride,     antiseptic    value    of, 
189 
sulphate,  action  of,  on  micrococcus 

pneumonise  crouposae,  40") 
antiseptic  power  of,  189 
Filters,  soluble,  in  analysis,  620 
Filtration  as  a  means  of  sterilizing,  58 
Finger-nails,  bacteria  found  under,  648 
Finkler  and  Prior,  spirillum  of,  602 
Fiocca's  bacillus,  547 

method  of  staining  spores,  32 
Fire  as  a  disinfectant,  212,  213 
Fish,  boiled,  as  a  culture  medium,  40 
Flagella  of  bacteria,  24,  119 
methods  of  staining,  32 
of  bacillus  amylovorus,  576 
campestris,  575 


INDEX. 


Flagella    of    bacillus     of    hog    cholera 

(Loffler's  staining),  504 
hyacinthi,  573 
cedeinatis  nialigni,  581 
redernatis  maligni,  No.  II.,  588 
solanacearum,  571 
of  swine  plague,  509 
of  symptomatic  anthrax,  586 
typhi  abdominalis,  437 
typhi  murium,  524 
proteus  vulgar  is,  550 
pseudomonas  Stewarti,  575 
spirillum  of  Finkler  and  Prior,  002 
Metschnikovi,  (504 
Obermeieri,  590 
Flasks,  Erlennieyer's,  for  liquid  media, 

60,  63 

Pasteur's,  63 
Sternberg's,  38,  66 

Fleas,  propagators  of  bubonic  plague,  504 
Fleckenkrankheit,  415 
Flesh-peptone  solution  as  a  culture  me- 
dium, 41 
gelatin,  preparation  of,  41 

Koch's,  use  of,  69 
Flesh  water  as  a  culture  medium,  40 
Flexner's  description  of  bacillus  dysen- 

terise,  568 
Fliigge's  micrococcus  septicus,  415 

directions   for  preparing  fermented 

milk,  139 

Foa  and  Scabia's  experiments  on  micro- 
coccus  pneumonise  crouposse,  342 
Foot-and-mouth  disease,  protective  inoc- 
ulations in,  318 

Formaldehyde  as  a  disinfectant,  204,  216 
Formalin,  disinfectant  power  of,  204 
use  of,  as  a  test  for  typhoid  bacilli, 

446 

Formic  acid  as  a  germicide,  183 
Formol,  disinfectant  power  of,  204 
Foth's  directions  for  preparing  mallein, 

320 
Fowl  cholera,  causes  of,  288 

bacillus  of,  action  of  chlorine 

on,  177 
bacillus  of,  action  of  germicides 

on,  501 

bacillus  of,  thermal  death-point 
of,  156 


Frankel's  method  of    cultivating    anae- 
robic bacteria,  82 
pneumococcus,  :)•']'.> 
researches  on  the  bacteria  of  the  soil, 

643 

Frankel-Gabbett  method  of  staining  ba- 
cillus tuberculosis,  471 
Fraukel   and   Simmonds,  multiplication 
of  typhoid  bacilli   in  the  spleen  after 
death,  433 

Freezing,    influence  of,  on  bacterial  vi- 
tality, 153 
French  commission,   report  of,   on  Fer- 

ran's  anticholeraic  inoculations,  301 
Freudenreich's   modification  of   Koch's 

plate  method,  71) 
Friedlander's  bacillus,  analysis  of,  124 

thermal  death-point  of,  155 
pneumococcus,  396 
method  of  staining  the  tubercle  ba- 
cillus, 30 

Fuchsin  as  a  stain,  25,  27 
Fuller's  method  of  determining  the  re- 
action of  nutrient  media,  50 
Furniture,  disinfectants  for,  213 

GABBETT'S  method  of  staining  bacillus 

tuberculosis,  30 

Gaffky's  explanation   of  the  failure  to 
find  Eberth's  bacillus  in  every  case  of 
typhoid  fever,  432 
Galbanum,  antiseptic  power  of,  203 
Gallic  acid,  action  of,  on  bacteria,  183 
Gas  developed  by  the  bacillus  of  Em- 
merich and  Weibel,  566 
by  bacillus  oedematis    nialigni, 

No.  II.,  588 

by  bacillus  piscicidus  agilis,  565 
by  bacillus  of  von  Dungern  (cap- 
sule), 562 

bacillus  (the)  in  its  relations  to  hu- 
man pathology,  589 
Gases,  action  of,  on  bacteria,  172 
Gaslight,  photomicrography  by,  109 
Gastric  juice,  germicidal  power  of,  658 
protective  by  its  acidity  against 

attacks  of  cholera,  600 
Gastro-enteritis    cholerica  of    chickens, 
symptoms    and    post-mortem    appear- 
ances, 605 


INDEX. 


687 


Gelatin  as  a  culture  medium,  41 

liquefaction  of,  by  bacteria,  135 
growth  of  bacillus  aerogenes  capsu- 

latus  on,  589 

of  bacillus  dysenteriae  on,  569 
of    bacillus    cedeiiiatis    rnaligni 

No.  II.  on,  588 

of  symptomatic  anthrax  on,  086 
of  bacillus  solanacearum  on.  072 
nutrient,  as  a  culture  medium,  42 
for  bacillus  diphtherias.  454 
for  bacillus  cedeiiiatis  maligni, 

582 

plates,  counting  the  colonies  on,  630 
plates  in  the  cultivation  of : 
bacillus  A  of  Booker,  555 
bacillus  acidiformans,  537 
bacillus  alvei,  556 
bacillus  anthracis,  425 
bacillus  of  Beck,  566 
bacillus  of  Belfonti  and  Pasca- 

rola,  508 

bacillus  capsulatus,  522 
bacillus  cavicida,  516 
bacillus  coli  communis,  531 
bacillus  crassus  sputigenus,  517 
bacillus  of  Emmerich  and  Wei- 

bel,  566 
bacillus  endocarditidis  griseus, 

555 

bacillus  enteritidis,  520 
bacillus  erysipelatos  suis,  512, 

513 

bacillus  of  Fiocca,  548 
bacillus  of  Friedlander,  397 
bacillus  gallinarum,  521 
bacillus  of  grouse  disease,  521 
bacillus  of  hog  cholera,  504,  509 
bacillus  lactis  aerogenes,  536 
bacillus  of  Laser,  524 
bacillus  of  Lucet,  526 
bacillus  of  Mereshkowsky,  565 
bacillus  of  Nicolaier  (capsule), 

560 

bacillus  piscicidus,  567 
bacillus  piscicidus  agilis,  565 
bacillus  of  purpura  hasmorrhagi- 

ca,  Tizzoni   and   Giovannini, 

558 
bacillus  pyocyaneus,  543 


Gelatin  plates  in  the  cultivation  of : 
bacillus  pyocyaneus  /3,  545 
bacillus   pyocyaneus   pericardi- 

tidis,  546 

bacillus  pyogenes  fcetidus,  518 
bacillus     septicaemias     hasmor- 

rhagicae,  500,  509 
bacillus  tetani,  579 
bacillus  typhi  abdominalis,  438 
bacillus  typhi  murium,  524 
bacillus   of  von  Dungern  (cap- 
sule, 562 

micrococcus  bothryogenus,  412 
inicrococcus  Manfredi,  413 
micrococcus  tetragenus,  411 
proteus  hominis  capsulatus,  519 
proteus  of  Karlinski,  552 
proteus  lethalis,  554 
proteus  mirabilis,  553 
proteus  septicus,  554 
proteus  vulgaris,  549 
proteus  Zenkeri,  554 
spirillum  choleras  asiaticae,  594 
spirillum  of  Finkler  and  Prior, 

603 

spirillum  Metschnikovi,  604 
spirillum  tyrogenum,  603 
staphylococcus  pyogenes  aureus, 

374 

streptococcus  pyogenes,  384 
in  water  analysis,  628 
stab  cultures  of : 

bacillus  A  of  Booker,  555 
bacillus  acidiformans,  537 
bacillus      acne      contagiosa    of 

horses,  557 
bacillus  alvei,  557 
bacillus  anthracis,  425 
bacillus  of  Babes  and  Oprescu, 

526 

bacillus  of  Beck,  566 
bacillus  of  Belfonti  and  Pasca- 

rola,  508 

bacillus  capsulatus,  522 
bacillus   cavicida    havaniensis, 

516 
bacillus  of  Cazal  and  Vaillard, 

525 

bacillus  of  cholera  in  ducks,  503 
bacillus  coli  communis,  531 


688 


INDEX. 


Gelatin,  stab  cultures  of  : 

bacillus  crassus  sputigenus,  518 
bacillus  cuniculicida  havanien- 

sis,  539 
bacillus  of  Emmerich  and  Wei- 

bel,  560 
bacillus  endocarditidis  capsula- 

tus,  556 
bacillus  endocarditidis  griseus, 

555 

bacillus  enteritidis,  520 
bacillus  erysipelatos  suis,  512 
bacillus  of  Friedlander,  397 
bacillus  gallinaruui,  521 
bacillus  gracilis  cadaveris,  560 
bacillus  of  grouse  disease,  521 
bacillus  of  hog  cholera,  504 
bacillus  hydrophilus  fuscus,  522 
bacillus  lactis  aerogenes,  536 
bacillus  of  Laser,  524 
bacillus  leporis  lethalis,  541 
bacillus  of  Lucet.  526 
bacillus  of  Nicolaier  (capsule), 

560 

bacillus  cedeinatis  maligni,  582 
bacillus  piscicidus,  567 
bacillus  of  purpura   hEemorrha- 

gica  (Babes),  558 
bacillus  of  purpura  hsemorrha- 

gica  (Kolb),  559 
bacillus  pyocyaneus,  543 
bacillus  pyocyaneus  ft,  545 
bacillus    pyocyaneus  pericardi- 

tidis,  546 

bacillus  pyogenes  fcetidus,  518 
bacillus  of  symptomatic  anthrax, 

586 

bacillus  tetani,  579 
bacillus  typhi  abdominalis,  439 
bacillus  typhi  murium,  524 
bacillus  of  von  Dungern,  562 
micrococcus  bothryogenes,  412 
micrococcus  Man  f red i,  413 
micrococcus  tetragenus,  41 1 
proteus  hominis  capsulatus,  519 
proteus  of  Karlinski,  552 
proteus  lethalis,  555 
proteus  mirabilis,  553 
proteus  septicus,  554 
proteus  vulgar  is,  549 


Gelatin  stab  cultures  of  : 

spirillum  cholerae  asiaticae,  595 
spirillus  of  Finkler  and  Prior, 

603 

spirillum  Metschnikovi,  605 
spirillum  tyrogenum,  603 
staphylococcus  pyogenes  aureus, 

374 

streptococcus  pyogenes,  384 
roll-tubes  for : 

bacillus  acidiformans,  537 
bacillus    cavicida   havaniensis, 

516 

bacillus  cuniculicida  havanien- 
sis, 539 

bacillus  grandis  cadaveris,  560 
bacillus  leporis  lethalis,  541 
stick  cultures,  staining  for,  36 
streak  culture  of  bacillus  of  Lucet, 

526 
culture    of    bacillus    of    swine 

plague,  Marseilles,  509 
Gentian  violet  as  a  stain,  25,  27 
Geranium,  antiseptic  power  of,  203 
Germicides,  definition  of,  164 

methods  of  determining  value  of,  166 
Germs  in  air,  number  of,  623 
Gessard  -on   the   pigments  produced   by 

bacillus  pyocyaneus,  134 
researches  on   bacillus  pyocyaneus, 

543 

Giant  cells  in  tuberculous  nodules, 468, 472 
in  lungs  from  injection  of  dead 

tubercle  bacilli,  485 
Glaeogenes  of  Cohn,  11 
Glanders,  animals  affected  by,  233 
bacillus  of  (B.  mallei),  489 
methods  by  which  infection  is  com- 
municated, 491 

test  for,  by  mallein,  152,  321,  494 
by  obtaining  pure  cultures  by 

inoculation,  493 
Glucose  in  culture  media,  46 

influence  of,  on  bacterial  growth,  128 
Glycerin,  action  of,  on  bacteria,  204 
Glycerin-agar,  preparation  of,  43 

bacillus  acidiformans,  growth  of,  on, 

538 

of  Babes  and  Oprescu,  626 
cuniculicida  havaniensis,  539 


INDEX. 


G89 


Glycerin-agar.  bacillus  hydrophilus  fus- 

cus,  523 
mallei,  490 
pestis,  563 

of  swine  plague.  Marseilles,  509 
tuberculosis,  476 
roll -tube  cultures  of  bacillus  cavi- 

cida  havaniensis,  517 
of  bacillus  cadaveris  in  hydro- 
gen atmospheres,  584 
Goat's  milk  immunized,  antitoxin  of,  357 
precipitation  of  antitoxin  from, 

358 

Gold  chloride  as  an  antiseptic,  190 
Gonococcus  of  Neisser,  226,  391 

biological  characters  of,  393 

discovery  of,  7 

multiplication  of,  in  leucocytes, 

253 

Graham  bread,  bacteriology  of,  672 
Grain's  method  of  staining,  29,  34 

solution  for  decolorizing  in  staining, 

28 

Grape  sugar  in  liquid  culture  media,  68 
growth    of    bacillus    coli    com- 

munis  in  solutions  of,  532 
Graveyards,  bacteria  in  the  soil  of,  643, 

(.4(5 

Gray-parrot  disease,  micrococcus  of,  419 
Ground-water  region  of  the  soil,  free  from 

bacteria,  644 

Guaiacol,  antiseptic  power  of,  204 
Guinea-pigs,  effects  of  injection  of  tuber- 
culous matter  into,  480 

ILEMATOCOCCUS  bovis  (Babes),  419 
Hsemoglobinuria  of  cows,  cocci  of,  419 
Haffkine's    anticholeraic     inoculations, 

302 

Hail,  number  of  bacteria  in,  632 
Haloid  elements,  action  of,  on  bacteria, 

172 

Hands,  disinfection  of  the,  216,  648 
Hanging-drop  cultures,  64 

of  typhoid   bacilli,   appearance 

of,  442 
Hansen's  discovery   of   the   bacillus  of 

leprosy,  7 
Hazen  and  White's  method  of  separating 

typhoid  bacilli  in  water  supplies,  445 
44 


Heat,  action  of,  on  anthrax  spores,  587 
attenuation  of  virulence  by,  131 
influence  of,  on    bacterial    growth, 
153 

on  bacillus  pestis,  564 
isolation  of  spores  by,  68 
as  a  sterilizer  of  culture  media,  52 
dry,  as  a  disinfectant,  212,  213 

influence  of,  on  bacterial  growth, 

154 

non-penetration  of,  154 
as  a  sterilizing  agency,  53 
moist,  influence  of,  on  bacterial  vi- 
tality, 154 
Heating,  discontinuous,  as  a  sterilizing 

measure,  53 

Heliostat  in  photomicrography,  107 
Helman's    tuberculin    from  potato  cul- 
tures, 363 

Helmholtz'S  experiments  opposed  to  spon- 
taneous generation,  4 
Hernialbumose,  262 

Herbivora,  susceptible  to  bacterial  injec- 
tion, 96 
to  anthrax,  234 
Hericourt  and  Richet,  experiments  of,  on 

bacillus  tuberculosis,  364,  365 
Hesse's  apparatus  for  the  bacteriological 

analysis  of  air,  618 

Heterogenesis    or    spontaneous    genera- 
tion, 5 

Heydenreich's  micrococcus,  415 
Hoffman's  experiments  on  the  preserva- 
tion of  putrescible  liquids,  5 
Hog    cholera,    bacillus    of    (Salmon  and 

Smith),  503 

prevalence,  symptoms  and  post- 
mortem appearances  of,  505, 
506 
protective  inoculations  against, 

322 
group   of  bacteria,  varieties  or 

species  '?  506,  507 
erysipelas,  bacillus  of,  511 

protective  inoculations  against, 

324 

symptoms  and  post-mortem  ap- 
pearances of,  514 

Holz's  method  of  searching  for  the  ty- 
phoid bacillus  in  water  supplies,  444 


690 


INDEX. 


Horse,  symptoms  of  glanders  in  the,  491 
immunization    of,    against  tetanus, 

360 
Horses,  bacillus  of  acne  contagiosa  of, 

557 

diplococcus  of  pneumonia  in,  418 
streptococcus  coryzae  contagiosae  of, 

418 

Hot-air  oven  for  sterilizing,  53 
Hot  Springs,  Ark.,  Malta  fever  from  the 

tropics  at,  420 
Hueppe's  apparatus  for  sterilizing  and 

coagulating  blood  serum,  58 
classification  of  bacteria,  12 
Hyacinth,  bacterial  disease  of  the,  574 
Hydrant  water,  number  of  bacteria  in, 

634 

Hydrocele  fluid  as  a  culture  medium,  40 
Hydrochloric  acid,  action  of,  on  micro- 
coccus  pneumoniae  crouposse, 
405 

as  a  decolorizer  in  staining,  28 
influence  of,  on  bacterial  growth, 

181 
Hydrofluoric   acid,    influence  of,  on  the 

tubercle  bacillus,  179 
Hydrogen  generator,  86 

in  the  cultivation  of  anaerobic  bac- 
teria, 82 

influence  of,  on  bacterial  growth,  173 
peroxide  as  a  disinfectant,  173 
Hydronaphthol,  antiseptic  power  of,  205 
Hydrophobia,    influence  of  temperature 
on  the  infectious  material  of,  156 
protective  inoculations  against,  332 
toxalbumin  of,  327 

Hydrosulphuric  acid,  a  bacterial  produc- 
tion, 141 
influence  of,  on  bacterial  growth, 

175 

Hydroxylamin,  antiseptic  power  of,  204 
Hypodermic  injection  of  cholera  vaccine, 

299 
syringe  for  inoculations,  97 

ICE,  number  of  bacteria  in,  633 

cream,  toxic  principle  in,  148 
Ichthyol,  antiseptic  power  of,  204 
Illicium,  antiseptic  power  of,  203 
Immunity  from  disease,  233 


Immunity,  acquired,  242 

alkalinity  of  the  blood  as  affecting, 

236 

dependent  on  antitoxins.  267 
influence  of  animal  temperature  ont 

235 

natural   agencies   by  which  neutral- 
ized, 240 
theories  of,  249 
to  anthrax  by  filtered  cultures  of  the 

bacillus,  428 
by  anthrax  toxin,  427 
to  cholera  by  a  previous  attack,  2!>7 
to  serpent  venom,  262 
to    tetanus   by   cultures   in  thymus 

bouillon,  359 
inherited,  360 

to  tuberculosis    by  attenuated  cul- 
tures, 485 
Immunization  of  swine  against  rouget, 

method  and  disadvantages  of,  515 
Impf tetanus  bacillus,  508 
Incubating  ovens,  88 
Indigestion,  sarcinae  in  stomach  of  per- 
sons suffering*  from,  662 
Indol,  antiseptic  power  of,  205 
production  of,  151 

by  the  cholera  spirillum,  598 
by  vibrios  resembling  the  chol- 
era spirillum,  607 
by  bacillus  septicaemias  hsemor- 
rhagicae   in  peptone  solution, 
501 
by    bacillus    of    swine    plague, 

Marseilles,  509 
by  capsule  bacillus  of  von  Dun- 

gern,  562 
not  produced  by  the  bacillus  of  hog 

cholera,  505 

test  for  the  typhoid  bacillus,  440 
Infection,  rapidity  of,  231 

by  inhalation,  102 
Infections,  mixed,  228 

secondary,  227 
Infectious  diseases,   chronic,  bacilli  in, 

467 
Influenza,  protective  inoculations  against, 

335 

bacillus,  discovery  of,  8 
morphology.  463 


INDEX. 


691 


Influenza,  bacillus,  pathogenesis,  464 
stains  for,  4i;:J 
toxin  of.  405 
vitality  of.  4(54 
in    horses,    protective    inoculations 

against.  335 

Inhalation,  infection  of  animals  by,  102 
Inoculation    experiments   with    bacillus 

pest  is,  503 
of  liquid  media,  03 

Inoculations,  protective,  in  cholera,  204 
Insects,  transmission   of  plant  diseases 

by,  570 
Intestinal  bacteria,  not  concerned  in  the 

physiology  of  digestion,  002 
mucousmembrane,  infection  through, 

230 
Intestine,  bacterial  injections  into.  W 

of  man,  bacteria  found  in,  000,  00.--J 
Involution  forms  of  bacilli,  23 

of  bacillus  of  symptomatic  an- 
thrax, 580 

Iodine,  action  of,  on  micrococcus  pneu- 
monias crouposae,  405 
influence  of,  on  bacterial  growth,  178 
trichloride  as  a  disinfectant,  178 
lodoform  as  an  antiseptic,  178 
lodoform-ether,    action    of,   on    bacillus 

tuberculosis.  179,  478 
lodol  without  germicidal  power,  179 
Issaeff 's  experiments  on  the  micrococcus 

pneumonias  crouposae,  342 
Izal,  disinfectant  power  of,  205 

JAHRESBE RIGHT  (the)  of  Baumgarten,  9 
Jequirity  solution  as  a  culture  medium,  47 
"Journal  of  Hygiene."  9 
"Journal    of     Pathology   and    Bacteri- 
ology, "  9 
Juniper,  antiseptic  power  of,  203 

KAESESPIRILLEX,  003 
Kidneys,  cultures  of  typhoid  bacilli  ob- 
tained from  the.  433,  430 
specially  affected  by  staphylococcus 

pyogenes  aureus,  370 
Kinyoun's   treatment   of    the  horse   for 

diphtheria  antitoxin,  310 
Kitasato,  bacteriological  work  of,  8 

distinguishing  biological  characters 
of  the  bacillus  of  influenza,  404 


Kitasato,  experiments  on  the  bacillus  of 

tetanus.  351 

results  with  tuberculin,  305 
Kitt's   micrococcus  of  bovine  mastitis, 

414 

Klebs  on  the  influence  of  Koch's  tuber- 
culin. 304 

method  of  obtaining  pure  cultures, 
^___6_Z_ 

Klebs-Loffler  bacillus  of  diphtheria,  449 
value  of,  for  diagnostic  purposes, 

451 

Klemperer's  experiments  on  cholera  im- 
munization. 308 
Koch's  apparatus  for  coagulating  blood 

serum,  58 

bacteriological  researches,  7 
decolorizing  solution,  28 
differentiation  of  species,  482 
discovery   of  the   tubercle  bacillus, 

407,  408 

experiment  showing  that  the  comma 
bacillus  will  infect  when  intro- 
duced in  a  living  condition  into 
the  intestine,  000 

experiments  on  the  thermal  death- 
point  of  bacteria,  154 
flesh-peptone  gelatin,  use  of,  09 
inoculation  syringe,  97 
method  of  determining  the  germi- 
cidal power  of  chemical  agents  on 
anthrax  spores,  108 
method  of  staining  flagella,  32 
plate  method,  74 

method,  modifications  of,  79 
method,  in  water  analysis,  029 
statement  of  the  action  of  sunlight 

on  the  tubercle  bacillus,  478 
steam  sterilizer,  55 
tuberculin,  151,  301 
production  of,  302 
experiments  favorable  to,  479 
typhoid  bacillus,  432 
vibrios  remotely  resembling  the  chol- 
era spirillum,  007 
views  of  the  interior  structure  of  the 

bacterial  cell.  118 
Kockel's  capsule  bacillus,  501 
Kolb's  bacillus  of  purpura  haeinorrhagicai 
559 


692 


INDEX. 


Kommabacilhis  der  cholera  asiatica,  593 

Kreibohm's  bacillus,  517 

Kruse's   modification    of    Koch's    plate 

method,  79 
Kuhne's  method  of  staining  bacteria  in 

tissues,  35 
silicate  jelly,  47 

staining  method  for  bacillus  mallei, 
400 

LACTIC  acid,  action  of,  on  bacteria,  182 

Lactose-litmus-agar,  47 

Lactose  and  litmus  for  detecting  typhoid 

bacilli,  445 

Lake  water,  number  of  bacteria  in,  633 
Lancet-shaped    micrococcus    (Talamon), 

398 

Lanolin,  antiseptic  power  of,  205 
Laundries,  bacteria  in  wash-water  from, 

649 

Lavender,  antiseptic  power  of,  203 
Lead  chloride  as  an  antiseptic,  190 

nitrate  as  an  antiseptic,  190 
Leprosy,  bacillus  of,  487 
Leptotrichese  of  Zopf,  general  characters, 

12,  19 
Leucocytes,  protective  against  bacterial 

invasion,  239 

multiplication  of  gonococci  in,  258 
Leuconostoc,  general  characters  of,  17 
Liborius'  method  of  cultivating  anaero- 
bic bacteria,  85 
Light,  attenuating  influence  of,  279 

influence  of,  on  bacterial  growth,  159 
action  of,  on  bacillus  tuberculosis, 

478 

for  photomicrography,  107 
Linen  underclothing,  bacteria  on,  649 
Liquefying  bacteria,  16 

in  stab  cultures,  71 

Liquid  media,  cultures  of  anaerobic  bac- 
teria in,  81 
Liquids,  non-evolution  of  bacteria  from 

the  surface  of,  614 

Lithium  chloride  as  an  antiseptic,  190 
Litmus,  in  culture  media,  46,  64 

action  of  bacillus  of  hog  cholera  on, 

504,  509 

Liver,    cultures  of    typhoid   bacilli  ob- 
tained from  the,  433,  436 


Lochial  discharge,   presence  of  bacteria 

in,  054 

Loftier 's  aniline-blue  solution,  29 
blood -serum  mixture,  48 
culture   medium   for  bacillus  diph- 
therias, 454 

method  of  staining  bacteria  in  tis- 
sues, 34 

of  staining  flagella,  '.}•! 
solution  for  staining  anthrax  bacilli, 

424 

for  staining  the  typhoid  bacil- 
lus, 4:;: 

Loretin,  disinfectant  power  of,  205 
Liibert's  researches  on  the  action  of  anti- 
septic  agents   on  staphylococcus  pyo- 
genes  aureus,  375 
Lustgarten's  bacillus,  494 
Lydton's   report   on  protective   inocula- 
tions in  Baden,  350 
Lymph,  germicidal  power  of,  236 
Lysol,  disinfectant  power  of,  205 

MACE,  antiseptic  power  of,  203 
Maffucci  's  conclusions  on  the  tuberculo- 
sis of  fowls,  484 
Maggiora's  researches  on  the  bacteria  of 

the  soil,  644 
Magnesium  light,  in  photomicrography, 

107 

Mail  matter,  disinfection  of,  214 
Mai  d'araigne"e,  micrococcus  of,  416 
Mai  de  pis,  micrococcus  of,  416 
Malic  acid  as  a  germicide,  183 
Malignant  oedema,  bacillus  of,  580 

pustule  in  man,  422,  428 
Mallein,  a  glycerin  extract  from  cultures 
of  the  bacillus  of  glanders,  318,  494 
as  a  test  for  glanders  in  horses,  321 
preparation  of,  in  Pasteur's  labora- 
tory, 319 

Foth's  method  of  preparing,  320 
Malnutrition,   influence  of,   on  develop- 
ment of  pus  cocci,  224 
Malta  fever,  micrococcus  of,  420 

Widal  reaction  in,  421 
Maminite    contagieuse,  micrococcus  of, 

417 

Manganese  protochlorite  as  an  antiseptic, 
190 


INDEX. 


693 


Marchand's  capsule  bacillus,  561 
Marjoram,  antiseptic  power  of,  203 
Marmier's  experiments  on  the  toxin  of 

anthrax,  430 
Marpmann's  method  for  the  detection  of 

pathogenic  bacteria  in  water,  040 
Marsh  gas,  a  bacterial  product,  141 
Mastitis  in  cows,  micrococcus  of.  417 

streptococci   of,    varieties  of   a 

single  species,  419 
gangrenous,    in   sheep,   micrococcus 

of,  410 

Matico,  antiseptic  power  of,  203 
Mauseseptikamiealmlicher  bacillus  (Eis- 

enberg),  515 

Measles,  second  attacks  of,  243 
Meat  infusions  as  culture  media,  40 
Meats,  bacteria  in,  070 
Meatus  urinarius  of  man,  bacteria  in, 

654 

Meconium,  free  from  bacteria,  660 
Media,  liquid,  cultures  in,  62 

characters  of  growth  in,  64 
solid,  cultures  in,  69 
Mediterranean  fever,  micrococcus  of,  420 
Melons,  bacterial  diseases  of,  576 
Merchandise,  disinfection  of,  214 
Mercuric  chloride  action  of,  on  anthrax 

spores,  587 

•action   of,  on  bacillus  tubercu- 
losis, 478 
action  of,  on  micrococcus  pneu- 

rnoniae  crouposae,  405 
as  a  disinfectant,  212,  213 
as  an  antiseptic,  190 
cyanide  as  a  disinfectant,  192 
iodide  as  an  antiseptic,  192 
Merismopedia,  general  characters  of,  17 

grouping  of,  in  tetrads,  22 
Mesenteric  glands,  cultures  of  typhoid 

bacilli  obtained  from,  433,  436 
Metachromatic  granules,  118 
Metacresol,  germicidal  power  of,  202 
Methane,     influence     of,    on     bacterial 

growth,  175 
Methylamine,  147,  :323 
Methylene  blue  as  a  stain,  25,  27 
Methyl-guanidin,  148 
Metschnikoff's    theory   of  phagocytosis, 
208,  256 


Mice,  subcutaneous  injection  of,  99 

field,  immune  to  mouse  septicaemia, 

515 

Mereshkowsky  's  suggested  meth- 
od of  destroying,  567 
Microbe  du  cholera  des  poules  (Pasteur), 

499 

du  pus  bleu,  542 

Micrococci,  general  characters,  17 
infrequent  in  the  soil,  645 
list  of    species   found   in    the    soil 

(Fulles),  646 
non-pathogenic,  list  of  species  found 

in  water,  638 
pathogenic,  list  of  species  found  in 

water,  638 

Micrococcus  askofornians  (Johne),  412 
of  Biskra  button,  415 
botbryogenus  (Rabe),  412 
of  bovine  mastitis,  414 

pneumonia,  414 
endocarditidis    rugatus    (Weichsel- 

baum),  416 

of  erysipelas  (Fehleisen),  382 
of  fowl  cholera,  thermal  death-point 

of,  150 

of  gangrenpus  mastitis  in  sheep,  416 
gonorrhceal,  391 

growth  of,  in  acid  media,  393 
thermal  death-point  of,  156 
of  Heydenreich,  415 
of  Manfredi,  413 
melitensis.  420 

of  myko-dermoids  of  the  horse,  412 
ovatus,  415 

Pasteuri  (Sternberg),  339,  398 
discovery  of,  7,  339,  398 
found  in  the  mouth.  652 
thermal  death-point  of,  155 
pneumoniae  crouposae,  339,  398 

crouposae,  biological   characters 

of,  403 

crouposae,  capsule  of,  402 
crouposae,  discovery  of,  7 
crouposae,  morphology  of,  402 
crouposae,  pathogenesis,  400 
crouposae,   thermal    death-point 

of,  404 

crouposae,     Belfonti's      experi- 
ments on,  341 


694 


INDEX. 


Micrococcus  pneumonias  crouposae,  Em- 
merich's experiments  on,  341 
crouposae,  Foa  and  Scabia's  ex- 
periments on,  342 
crouposae,  Issaeff's  experiments 

on,  341 

crouposae,    Kleniperer's    experi- 
ments on,  342 
crouposae  in  endocarditis,  ulcer- 

ative,  401 

crouposae  in  meningitis,  400 
crouposae  in  otitis  media.  390, 

402 
of  progressive  granuloma  formation, 

413 

pyogenes  tenuis,  382 
of  sputum  septicaemia  (Frankel),  398 
tetragenus,  411 

action  of  chlorine  on,  177 
thermal  death-point  of,  155 
urese,  140 

liquefaciens,  140 
Micromillimetre  (the),  20 
Microzyma  bombycis,  415 
Middle-ear  disease,  bacillus  pyocyaneus 

in,  545 
Milk,  bacteria  in,  232,  663 

number  of  present,  669 
fermented,  139 
immunization  by,  263 
of  immunized  goats,  antitoxic,  357 
toxic  principle  in,  148 
of  healthy  women,  bacteria  in,  669 
of  cows  affected  with  chronic  masti- 
tis, 417 
of  tuberculous  cows,  infective,  482, 

485,  669 
as  a  culture  medium,  39 

for  bacillus  A  of  Booker,  555 
for  bacillus  aerogenes  capsulatus, 

589 

for  bacillus  alvei,  557 
for  bacillus  coli  communis,  532 
for  bacillus  diphtheriae,  455 
for  bacillus  dysenteriae,  569 
for  bacillus  of  Fiocca,  548 
for  bacillus  of  hog  cholera,  504, 

509 

for  bacillus  of  Laser,  524 
for  bacillus  piscicidus,  567 


Milk  as  a  culture  medium  for  bacillus 

piscicidus  agilis,  565 
for  bacillus  pyocyaneus,  543 
for  bacillus  pyocyaneus  pericar- 

ditidis,  547 

for  bacillus  solanacearum,  572 
for  bacillus  tenuis   sputigenus, 

523 
for  bacillus  typhi  abdominalis, 

439 

for  bacillus  typhi  rnurium,  524 
for  bacillus  of  vonDungern  (cap- 
sule), 562 
for     micrococcus      pneumonias 

crouposae,  404 
for  spirillum  cholera  asiaticae, 

596 

for  spirillum  Metschnikovi,  605 
Milzbrand,  bacillus  of,  422 
Mint,  antiseptic  power  of,  203 
Miquel's  aeroscope,  615 

later  methods  of  air  analysis,  618 
tables  of  antiseptic  values,  186 
Mironoff's  experiments  against  strepto- 
cocci c  infection,  346 
Mitylotoxin,  148 
Mixed  infections,  228 
Moist  chamber  for  drop  cultures,  65 
Moitessier's  gas-pressure  regulator,  88 
Moller's  method  of  staining  spore  prepa- 
rations, 32 
Monkeys,  susceptible  to  the  infection  of 

bubonic  plague,  564 

Monochlorophenol  as  a  disinfectant,  179 
Morphia  hydrochlorate  as  an  antiseptic, 

193 
Morphological  classification  of  bacteria, 

12 
Morphology  of  bacteria,  general  account, 

20 
insufficient  to  differentiate  species, 

20 
Mosny,     experiments     on     micrococcus 

pneumonias  crouposae,  342 
Motile  bacteria,  16 
Moulds,  spores  of,  in  atmospheric  dust, 

614 

Mouse  septicaemia,  symptoms  and  post- 
mortem appearances  of,  515 
Mouth,  bacteria  found  in  the,  651,  656 


INDEX. 


695 


Mucorini  of  Nageli,  11 

Mucus,  gerrnicidal  power  of,  210 

Miiiicke's  thermo-regalator.  92 

Muscarini,  147 

Mushrooms,  toxic  principle  of,  147 

Mussels,  toxic  principle  in,  148 

Mustard,  antiseptic  power  of,  203 

oil  of,  antiseptic  power  of,  200 
Mycelium  of  moulds,  614 
Mycoderma,  64 

acetic  acid  produced  by,  137 
Mycophylaxin,  265 
Mykoprotein,  124 
Myrtle,  antiseptic  value  of,  203 

NAOELI'S  classification  of  bacteria,  11 
Nails,  finger,  surgical  care  of,  217 
IS" ail-shaped  growth  of  stab  cultures,  397 
Naphthol,  disinfectant  power  of,  205 
Nasal  catarrh,  pus  cocci  in,  390 
Natural  culture  media,  37 
Neapolitan  fever,  micrococcus  of,  420 
Negro,    insusceptibility    of,    to    yellow 

fever,  234 
Neisser's  gonococcus,  226 

discovery  of,  7 

stain  for  bacillus  diphtherias,  457 
double   staining    of  spore  prepara- 
tions, 31 

Nematogenes  of  Colin,  11 
Neuridin,  146 
Neurin,  147 

Nickel  sulphate  as  an  antiseptic,  193 
Nicolaier's  capsule  bacillus,  560 

discovery  of  the  tetanus  bacillus,  8 
Nitrates,  reduction  of,  144 
Nitric  acid  as  a  decolorizer  in  staining, 

28 
influence  of,  on  bacterial  growth, 

181 
action  of,  on  micrococcus  pneu- 

monise  crouposse,  405 
Nitrification  produced  by  bacteria,  144 
Nitrogen  necessary  for  bacterial  growth, 

126 
dioxide,   influence   of,  on  bacterial 

growth,  175 
Nitrous  acid  as  a  test  for  indol,  151 

influence  of,  on  bacterial  growth, 
181 


Nitrous  oxide,  influence  of,  on  bacterial 

growth,  175 
Non-liquefying  bacteria,  16 

characters    of    growth    in    stab 

cultures,  70 

Non-motile  bacteria,  16 
Non-pathogenic  bacteria  from  the  mouth, 

656 

from  the  nose,  656 
list  of  species  from  faeces,  663 
Nose,  bacteria  found  in  the,  650,  656 
Nosema  bombycis,  415 
Nosophen,  179 
Nuclein,   the  gerrnicidal  constituent  of 

blood  serum,  239 

Nucleins,  germicidal  power  of,  210 
Nutmeg,  antiseptic  power  of,  203 
Nutrient  agar  (see  Agar,  nutrient) 
gelatin  (see  Gelatin,  nutrient) 
NuttalTs  method  of  estimating  bacilli  in 
tuberculous  sputum,  479 

OIL  light,  Borden's  method  of  photograph- 
ing by,  111 
Oleic  acid,    influence    of,   on    bacterial 

growth,  183 
Olive  oil,  no  influence  on  anthrax  spores, 

206 

Onion,  antiseptic  power  of,  203 
Opopanax,  antiseptic  power  of,  203 
Orange  of  Portugal,  antiseptic  power  of, 

203 

Origanum,  antiseptic  power  of,  203 
Orthochromatic  photographic  plates,  105, 

113 

Orthocresol,  germicidal  power  of,  202 
Orthophenol,  germicidal  power  of,  197 
Ose,  74 
Osmic  acid,    fatal    to    anthrax    spores, 

181 
Osteomyelitis,  micrococcus  of,  373 

thermal  death-point  of,  375,  378 
Otitis  "media,    micrococcus    pneumonias 

crouposse  in,  402 
pus  cocci  present  in,  389 
Oxalic  acid,  action  of,  on  bacteria,  182 
Oxychinaseptol.  antiseptic  power  of,  202 
Oxycyanide  of  mercury  as  a  disinfectant, 

192 
Oxygen,  as  a  disinfecting  agent,  172 


696 


INDEX. 


Oxygen,  essential  for  certain  species  of 

bacteria,  172  , 

influence  of,  on  pigment  production, 

133 

on  spore  formation,  12o 
nascent,  as  a  germicidal  agent,  172 

PANHISTOPHYTON  ovatum,  415 
Paracresol,  germicidal  power  of,  202 
Paralysis,  local,  caused  by   diphtheritic 

inoculations,  452 
Parasites,  strict,  221 
Parasitic  bacteria,  15 
Parietti's  method  of  separating  typhoid 

bacilli  from  water  supplies,  444 
Park  and   Beebe's  observations  on  the 
disappearance  of  the  Loftier  bacillus  in 
cases  of  diphtheria,  461 
Parrot  disease,  micrococcus  of,  419 
Pasteur's  bacteriological  researches,  7 
cholera  des  poules,  288 
demonstration  of  living  organisms  in 

the  atmosphere,  615 
discovery  of    the  causation    of  pe"- 

brine,  6 
experiments  on   the  attenuation  of 

virus,   130 

on  the  attenuation  of  the  mi- 
crobe of  fowl  cholera,  502 
on  the  etiology  of  rouget,  516 
on  the  preservation  of  putresci- 

ble  liquids,  5 
flasks  for  liquid  media,  63 
inoculations  of  rabbits  with  hydro- 
phobic  virus,  99 
solution,  40,  126 
tubes    for    the    collection  of  water 

samples,  627 

views  on  the  origin  of  epidemics,  290 
on  rabies,  328 

on  the  influence  of  temperature 
on  the  destruction  of  organ- 
isms, 5 

Pasteur-Chamberland  filter,  60 
Pasteur  Institute,   production   of    diph- 
theria antitoxin  at,  316 
of  Naples,  statistics  of,  331 
of  Palermo,  statistics  of,  331 
of  Paris,  statistics  of,  331 
of  Turin,  statistics  of,  331 


Patchouly,  antiseptic  power  of,  203 
Pathogenic  anaerobic  bacteria,  80 

bacteria,    list   of   species   found   in 

faeces,  6(5:; 
in  milk,  068 
in  the  mouth,  652,  656 
in  the  nose,  <>.~,i; 
in  the  soil,  duration  of  vitality 

of.  (i4o 
in  water,  6:56 

in  water,  detection  of,  640 
organisms,  14 

power  of  saprophytes  dependent  on 
quantity  and  age  of  cultures  and 
site  of  the  inoculation,  529 
spirilla,  590 

Paulsen's  capsule  bacillus,  561 
Pear  blight,  575 

Pe'brine,  silkworms  infected  with,  415 
causation  of,  discovered  by  Pasteur, 

6 

not  a  bacterial  disease  ?  415 
Pendesche  Geschwur,  415 
Pepper,  antiseptic  power  of,  203 
Peptone,  a  product  of  bacterial  action, 

142 

Peptotoxin,  148 

Peritoneum,  injections  into,  98 
Peritonitis,  bacillus  coli  commuuis  in, 

535 

Person,  disinfection  of  the,  213 
Petermann's    experiments   with   filtered 

anthrax  cultures,  428 
Petri  dishes,  76 

use  of,  in  air  analyses,  617 

use  of,  in  water  analyses,  630, 

631 

Petri 's  sand  filter  for  air  analyses,  620 
Petroleum   lamps   for  incubating  ovens, 

95 
Pfeiffer's  capsule  bacillus,  561 

evidence  of  his  bacillus  being  the 

specific  cause  of  influenza,  464 
researches  into  the  toxins  of  cholera 

cultures,  599 
views  on   the  toxin  of  the  typhoid 

bacillus,  367 

Pfeiffer  and  Issaeff's  method  of  distin- 
guishing the  cholera  spirillum  from 
other  vibrios,  609 


INDEX. 


097 


Pfuhl's  experiments  with  tuberculin,  365 
Phagocytosis,  255,  267 

Metschnikoff's  theory  of,  208,  256 
Phenol,    produced    by   bacillus  septicse- 

mise  haemorrhagicse,  501 
by    bacillus    of    swine  plague, 

Marseilles,  509 
not    produced    by   bacillus    of    hog 

cholera,  505 

Phenolphthalein,  to  determine  the  reac- 
tion of  media,  49 
Philippine  Islands,  Malta  fever  in  the, 

420 
Phosphorescence  by  bacteria,  145 

an    indication   that  vibrios  are  not 

the  cholera  spirillum,  608,  609 
Phosphoric  acid,  influence  of,  on  the  ty- 
phoid bacillus,  181 
Photomicrographs  of  bacteria    103 

amplification  of,  106 
Photomicrography,  apparatus  for,  106 
Physicians,  interest  of,  in  bacteriological 

work,  6 
Pigment,  production  of,  by  bacteria,  133, 

135 
by  staphylococcus  pyogenes  au- 

reus    374 
Pitfield-Muir  method  of  staining  flagella, 

33 

Plant  diseases   bacteria  of,  571 
Platinum  bichloride  a^an  antiseptic,  193 
loop  for  inoculations,  63 
needle  for  inoculations,  65,  69 
Plectridium,  spore  formation  in,  122 
Pleuropneumonia    of    cattle,    protective 

inoculations  against.  336 
Pneumonia,  causation  of,  241 
the  micrococcus  of,  339 
percentages  of  relapses  in,  340 
protective  inoculations  against,  338 
Pneumotoxin,  261,  340 
Polar  granules,  118 

Porcelain,    unglazed,   as  a  filtering  me- 
dium, 59 
Post-mortem  examination  of  inoculated 

animals,  101 
Potash    soap,    action    of,    on    bacterial 

growth,  185 

Potassium  hydroxide,    influence  of,    on 
bacteria,  183 


Potassium  nitrate  in  culture  media,  46 
permanganate  as  an  antiseptic,  194 
action  of,  on  micrococcus  pneu- 

moniab  crouposse,  405 
salts  as  antiseptics    193 
Potato  as  a  culture  medium,  40,  48,  78 
for  bacillus  A  of  Booker,  555 
for  bacillus  acidiformans,  538 
for  bacillus  aerogenes  capsulatus, 

589 

for  bacillus  alvei,  557 
for  bacillus  anthracis   426 
for  bacillus  of  Babes  and  Opres- 

cu,  526 

for  bacillus  capsulatus,  522 
for  bacillus  cavicida  havanien- 

sis,  516 
for  bacillus  of  Cazal  and  Vail- 

lard,  525 
for  bacillus  of  cholera  in  ducks, 

603 

for  bacillus  coli  communis    531 
for  bacillus  crassus  sputigenus, 

518 
for  bacillus  cuniculicida  havani- 

ensis,  539 

for  bacillus  diphtherias,  454 
for  bacillus  dysenterise.  569 
for  bacillus  of  Emmerich  and 

Weibel,  566 
for   bacillus  endocarditidis  gri- 

seus,  556 

for  bacillus  enteritidis,  520 
for  bacillus  of  Fiocca,  548 
for  bacillus  of  Friedlander,  397 
for  bacillus  gallinarum,  521 
for  bacillus  gracilis  cadaveris, 

554 
for  bacillus  of  hog  cholera   504, 

509 
for  bacillus  hydrophilus  fuscus, 

523 

for  bacillus  lactis  aerogenes  536 
for  bacillus  of  Laser,  524 
for  bacillus  leporis  lethalis,  541 
for  bacillus  of  Lucet,  527 
for  bacillus  mallei,  491 
for  bacillus  of  Nicolaier  (cap- 
sule), 560 
for  bacillus  pestis,  563 


698 


INDEX. 


Potato  as  a  culture  medium  for  bacillus 

piscicidus,  567 

for  bacillus  piscicidus  agilis,  565 
for  bacillus  of  purpura  hsemor- 

rhagica  (Babes),  558 
for  bacillus  of  purpura  hsemor- 

rhagica  (Kolb).  559 
for  bacillus  of  purpura  hsemor- 

rhagica  (Tizzoni  and  Giovan- 

nani),  658 

for  bacillus  pyocyaneus,  543 
for  bacillus  pyocyaneus  ft,  645 
for  bacillus  pyocyaneus  pericar- 

ditidis,  546 
for  bacillus   pyogenes   fcetidus, 

618 
for  bacillus  septicsemise  hsemor- 

rhagicse,  500,  509 
for  bacillus  solanacearum,  572 
for    bacillus    of    swine  plague, 

Marseilles,  509 
for  bacillus   tenuis  sputigenus, 

523 

for  bacillus  tuberculosis,  477 
for  bacillus  typhi  abdominalis, 

439 

for  bacillus  typhi  murium,  524 
for  bacillus  of  von  Dungern,  562 
for    micrococcus    bothryogenus, 

413 

for  micrococcus  Manfredi,  413 
for      micrococcus      pneumoniae 

crouposse,  403 

for  micrococcus  tetragenus,  412 
for  proteus  hominis  capsulatus, 

518 

for  proteus  of  Karlinski,  552 
for  proteus  lethalis,  555 
for  proteus  septicus,  554 
for  proteus  vulgaris,  549 
for  spirillum  cholerae  asiaticse, 

596 
for    spirillum    of    Finkler   and 

Prior,  603 
for  spirillum  of  Metschnikovi, 

605 

for  spirillum  tyrogenum,  604 
for  staphylococcus  pyogenes  au- 

reus,  375 
rot,  symptoms  of,  571 


Pregl's  method  of  staining  tubercle  ba- 
cilli in  tissues,  36 

Pressure,  influence  of,  on  growth  of  bac- 
teria, 163 

Privy  vaults,  disinfection  of,  217 
Protection  (standard  of)  in  inoculation 

of  an ti tetanic  serum,  356 
Protective  inoculations  in  tuberculosis, 

360 
against    typhoid    fever    in    the 

British  army,  369 

Proteus  capsulatus  septicus  (Banti),  520 
Hauseri     (Levy)     from    sour  yeast, 

551 

of     Karlinski,     probably    identical 
with  proteus  vulgaris  (Hauser),  552 
lethalis,  554 
bei    Lungengangran    des    Menschen 

(Babes),  654 
mirabilis,  swarming  islands  of,  552, 

553 

septicus,  554 
vulgaris  (Hauser),  548 

in  cholera  infantum,  550 
influence  of,  on  the  virulence  of 

anthrax  cultures,  587 
instanced  as  illustrating  varia- 
tions in  the  pathogenic  power 
of  saprophytes,  529 
Zenkeri,  554 

Prudden's    failure    to    find    the    Klebs- 
Loffler  bacillus  in  pseudo-membranous 
inflammations,  450 
Pseudo-diphtheria  bacilli,  461 

non  pathogenic.  450,  456 
attenuations    of    bacillus  diph- 
theria, 260 

Pseudo-influenza    bacillus    in    broncho- 
pneumonia,  466 
Pseudomonas  campestris,  575 
hyacinthi  (Wakker),  573 
Stewarti  (Smith),  575 
Ptomains,  133, 146 
Puerperal  fever  caused  by  streptococcus 

pyogenes,  387,  654 
self-infection  possible,  655,  656 
Pulmonary  mucous  membrane,  infection 

through,  230 

Pulmonic  anthrax,  422,  428 
Pure  cultures  on  gelatin  plates,  72 


INDEX. 


699 


Purpura  haeinorrhagica,  bacillus  of  Babes, 

558 

bacillus  of  Kolb,  559 
bacillus  of  Tizzoni  and  Giovan- 

nini,  558 
Pus,  formation  of,  223 

independent  of  bacteria,  371 
of  acute  abscess   micrococci  of,  14 
production   of.  by   typhoid  bacilli 

448 

cocci  found  in  healthy  eyes,  650 
found  in  the  mouth,  652 
present    in    diphtheritic    mem 

branes,  449 
on  inflamed  mucous  membranes, 

389 
sterilized   by  heat  give  rise  to 

pus  formation,  371 
Putrefaction,  141,  665 

anaerobic  bacteria  active  in,  142 

products  of,  142 

in    small    intestine     restrained    by 

bacteria,  662 
Putrescin    147 

Pyelonephritis,  proteus  vulgaris  in,  551 
Pyocyanin,  134,  543 
Pyogenic  bacteria,  371 
Pyoktanin,  germicidal  power  of,  197 
Pyrogallic  acid  in  the  cultivation  of  an 
aerobic  bacteria,  85 

QUARTER  evil,  347,  586 

Quinine,  salts  of,  as  antiseptics,  194 

RABBITS,  bacillus  of  intestinal  diphtheria 

in,  459 
inoculations  of   with  the  diphtheria 

bacillus,  452,  455 
with  hydrophobic  virus,  99 
with  tuberculous  matter,  481 
Race  immunity  to  disease.  234 
Rags,  disinfection  of,  214 
Railway  cars,  disinfection  of,  214 
Rain-water,  number  of  bacteria  in,  632 
Ranvier's  moist  chamber,  65 
Rats  as  propagators  of  bubonic  plague, 

564 

Rauschbrand,  347 
Rauschbrandbacillus.  585 

anon -virulent  variety  of  (Klein),  587 


Reaction  of  culture  media,  127 
Recurrens  spirochaete,  590 
Red  rays  of  spectrum  non  germicidal,  161 
Reichert's  ther mo-regulator,  90 
Relapsing  fever,  a  disease  of  man,  233 
spirillum  of,  590 
transmission  of,  by  inoculation 

with  infected  blood,  591 
Retention  theory  of  immunity,  250 
Rhinoscleroma,  bacillus  of,  496 
Rhorbeck's  thermo-regulator.  88 
Ricin,  immunity  against,  262,  265,  267 
Rinderpest,  influence  of  temperature  on 

the  infectious  material,  156 
protective  inoculations  against,  343 
Rinderseuche,  287 

River  water,  number  of  bacteria  in,  632 
Rose,  antiseptic  power  of,  203 
Rosemary,  antiseptic  power  of,  203 
Rotzbacillus  (see  Bacillus  mallei),  489 
Rouget,  287 

bacillus  of,  511 

Koch's  experiments  on  the  etiology 

of,  515 

protective  inoculations  against,  324 
Roux's  attenuated  varieties  of  diphtheria 

bacillus   309 

method  of  producing  diphtheria  anti- 
toxin, 316 

incubator  and  thermo-regulator,  95 
Roux  and  Yersin,  inoculation  of  the  ba- 
cillus of  diphtheria  into  rabbits, 
452 

study  of  the  pseudo-diphtheritic  ba- 
cilli, 456 

SACCHAROMYCETES  of  Nageli,  11 
Sach's  classification  of  bacteria    11 
Sage,  antiseptic  power  of,  203 
Salicylic  acid,  action  of.  on  bacillus  tu- 
berculosis, 478 
germicidal  action  of,  182 
action  of.  on  micrococcus  pneu- 
monias crouposas,  405 
Saliva,  peptonizing  action  of,  653 

prejudicial  to  the  action  of  certain 

pathogenic  organisms   653 
Salmon's  immunizing  experiments,  290 
Salmonson's  culture  method,  79 
tube,  82 


700 


INDEX. 


Salt  meat,  bacteria  in,  671 

saturated  solution  of,  inefficient  as  a 

germicide,  573 
Salts,  antiseptic  and  germicidal  value  of, 

158 
Sanarelli's  water  vibrios  resembling  the 

cholera  spirillum,  607 
Sandalwood,  antiseptic  power  of,  203 
Sand  filtration  in  air  analysis,  620 
Saprin,  147 

Saprol,  disinfectant  power  of   206 
Saprophytes,  611 

conditions  favorable  to  their  growth, 

613 

in  air,  list  of,  624 

Saprophytic    bacilli    which    are    patho- 
genic, 528 
bacteria,  15 

Sarcina,  general  characters  of,  17 
grouping  of,  22 
in  stomach  of  persons  suffering  from 

indigestion,  662 
aurantiaca,   thermal  death-point  of, 

155 

lutea,  thermal  death-point  of,  155 
Sassafras,  antiseptic  power  of,  203 
Savin,  antiseptic  power  of,  203 
Sawizky  on  the  vitality  of  dried  tuber- 
culous sputum,  479 
Scarlet  fever,  second  attacks  of,  243 
Scheinfaden,  120 
Schizomycetes  of  Nageli,  11 
Schoolrooms,  bacteriology  of  the  air  of, 

625 
Schroder  and  Van  Dusch,  use  of  cotton 

as  a  filter  by,  4 
Schuhanka,   protective    inoculations    in 

Salzburg   350 
Schulze's  (Franz)  experiments  opposed  to 

spontaneous  generation.  4 
Schiitz's   diplococcus   of  pneumonia  in 

horses  418 

Schwann's  experiments  opposed  to  spon- 
taneous generation,  4 
Schweinerothlauf,  287 
Schweineseuche,  287,  344 

(Loffler  and  Schtitz),  343 
Sea  water,   number  of  bacteria  in,  637, 

641 
Secondary  infections,  227 


Septicaemia,  definition  and  general  char- 
acters of,  498 
produced   by   saprophytic    bacteria, 

528,  529 

Septicsemic    bacteria,    attenuated   varie- 
ties in  the  mouth  and  air  passages  of 
certain  animals,  503 
Serpent   venom,   immunization   against, 

262 
Serum-albumin,  germicidal  power  of,  210, 

238 
Serum  of  blood,  germicidal   action  of, 

236,  257 

immunizing  value  of,  313 
Sevestre's  observations  on  the  disappear- 
ance of  the  Loffler  bacillus  after  diph- 
theria, 460 

Sewer  water,  number  of  bacteria  in,  634 
Shakespeare's  report  on  anticholeraic  in- 
oculations, 300 

Sheep,  micrococcus  of  gangrenous  masti- 
tis in,  416 
Sheep-pox,    influence  of  temperature  on 

the  infectious  material  of,  156 
Ships,  disinfection  of,  214 
Siberian  plague,  422 
Silicate  jelly  as  a  culture  medium,  47 
Silkworms  infected  with  "la  flacherie," 

415 

with  pe"brine,  415 
Silver  chloride  as  an  antiseptic,  194 

nitrate  as  an  antiseptic,  194 
Sjobring's  method  of  demonstrating  the 

structure  of  bacterial  cells,  117 
Skatol,  no  germicidal  power,  206 
Skin,  abscesses  caused  by  staphylococcus 

epidermidis  albus,  372 
human,  bacterial  flora  of,  648 

list  of  species  found  on  the,  649 
infection  through  the,  229 
Sleskin's  silicate  jelly,  47 
Smallpox,  heat  as  a  disinfectant  for,  154 

second  attacks  of,  243 
Smear  preparations,  26 
Smith  (Erwin   F.)  quoted  on  bacterial 

diseases  of  plants,  571 
Smith  (Theobald),  fermentation  tube  of, 

68 

views  on  the  hog  ckolera  group   of 
bacteria,  506 


INDEX. 


701 


Smith  (Theobald),  detection  of  colon  ba- 
cilli in  waters,  535 
identification  of  colon  and  typhoid 

bacilli  in  water  supplies,  445 
Smoke,  antiseptic  power  of,  206 

tobacco,  antiseptic  power  of,  207 
Snow,  number  of  bacteria  in,  632 
Sodium  borate  as  an  antiseptic,  195 
carbonate  as  an  antiseptic,  195 
chloride  as  an  antiseptic,  195 
in  culture  media,  -Mi 
influence  of,  on  bacterial  growth, 

128 
hydroxide,  influence  of,  on  bacterial 

growth,  i84 

hyposulphite  as  an  antiseptic,  195 
sulphite  as  an  antiseptic,  195 
Soil  bacceria,  642 
Solid  culture  media,  41 
Solidification  of  blood  serum,  57 
Sour  dough,  bacteria  of,  672 
Sozoiodol  acid  as  a  disinfectant,  179 
Spallanzani's  sterilization  of  putrescible 

liquids,  4 

Species  not  differentiated  by  morpholog- 
ical characters  alone,  13 
Sperulina  (Hueppe),  general  characters  of, 

18 

Spirilla,  general  characters  of,  18 
of  Bauingarten,  13 
list  of  species  found  in  water,  639 
movements  of,  119 
pathogenic,  590 
resembling  the  spirillum  of  Asiatic 

cholera,  606 

Spirillum    anserum,    from   blood  of  af- 
fected geese,  592 
cholera?  asiaticae,  292,  593 

asiaticae,  attenuation  of,  by  con- 
tinuous cultivation,  601 
asiaticie,   biological   characters, 

594 

asiaticae,  influence  of  disinfect- 
ing agents  on,  597 
asiaticae    influence     of     gastric 

juice  on,  658 
asiaticae,  influence  of  light  on, 

162 

asiaticae,  influence  of  tempera- 
ture on.  610 


Spirillum  choleras  asiaticae  occasionally 
present  in  healthy  persons,  610 
asiaticae,  phosphorescence  occa- 
sionally found  in,  609 
asiaticae,  thermal  death-point  of, 

155 
asiaticae,  vitality  of,   in  faeces, 

610 
asiaticae,      vitality      of,     when 

buried  in  the  soil,  64"> 
asiaticae,  vitality  of,   in  waters 

of  various  characters,  637 
of  Ehrenberg,  3,  10 
of  Finkler  and  Prior,  602 
pathogenesis,  603 
thermal  death-point  of,  155 
Metschnikovi,  604 

pathogenesis,  605 

murinum  (Russell)  remotely  resem- 
bling the  spirillum  of  cholera,  606 
Obermeieri,  in  the  blood  during  the 
febrile  paroxysms  of  relapsing 
fever,  590 
pathogenesis,  591 
tyrogenum  (Deneke),  603 

thermal  death-point  of,  155 
Spirochaete,  general  characters,  18 
anserina  (Sakharoff),  592 
of  Ehrenberg,  3,  10 
Obermeieri,  590 

discovery  of,  7 
Spleen,  influence  of,  on  the  spirillum  of 

relapsing  fever,  592 
pure  cultures  of  the  typhoid  bacillus 

from  the,  433 
Splenic  fever,  422 

Spontaneous  generation  proved   errone- 
ous, 4 
Spores,  16 

action  of  acetic  acid  on,  182 

endogenous,  120 

germination  of,  122 

influence  of  oxygen  and  temperature 

on,  123 

isolation  of,  by  heat,  68 
resisting  powers  of,  5,  157 
restraining  influence  of  temperature 

on,  166 

staining  of,  31 
thermal  death-point  of,  53,  473 


702 


INDEX. 


Spores,  vitality  of,  121,  122 
Spores  formed  by  : 

bacillus  alvei,  556 
bacillus  anthracis,  424,  426 
bacillus    anthracis,    multiplica- 
tion of,  429 
bacillus   anthracis,  survival  of, 

in  various  media,  430 
bacillus    anthracis,    exceptions 

to,  426 

bacillus  crassus  sputigenus,  517 
bacillus  cedematis  maligni,  582 
bacillus  piscicidus  agilis,  565 
bacillus  of  symptomatic  anthrax, 

586,  587 

bacillus  tetani,  578 
bacillus  tetani,  influence  of  heat 

and  germicides  on,  580 
Spores  not  formed  by  or  not  observed  in : 
bacillus  acidi  formans,  538 
bacillus    aerogenes    capsulatus, 

589 

bacillus  amylovorus,  576 
bacillus  of  Babes  and  Oprescu, 

526 

bacilus  of  Beck,  566 
bacillus  coli  communis,  530 
bacillus  cuniculicida  havanien- 

sis,  540 
bacillus  of  Emmerich  and  Wei- 

bel,  566 

bacillus  erysipelatos  suis,  513 
bacillus  of  Fiocca.  547 
bacillus  gracilis  cadaver  is,  560 
bacillus  hyacinthi,  573 
bacillus  lactis  aerogenes,  536 
bacillus  of  Laser,  524 
bacillus  leprse,  488 
bacillus  mallei,  489,  490 
bacillus  of  Mereshkowsky,  565 
bacillus  of  Nicolaier  (capsule), 

560 
bacillus  cedematis  maligni  No. 

II.,  588 

bacillus  pestis,  563 
bacillus  piscicidus,  567 
bacillus    of     purpura     hsemor- 

rhagica  (Babes),  558 
bacillus     of    purpura    haemor- 

rhagica  (Kolb),  559 


Spores  not  formed  by  or  not  observed  in : 
bacillus  pyocyaueus.  543 
bacillus  pyocyaneus    pericardi- 

tidis.  547 

bacillus  solanacearum,  571 
bacillus  tracheiphilus,  577 
bacillus  tuberculosis,  472,  478 
bacillus  of  von  Dungern  (cap- 
sule), 562 

proteus  lethalis,  554 
proteus  Zenkeri,  554 
spirillum  cholerae  asiaticse,  594 
spirillum  of  Finkler  and  Prior, 

603 

spirillum  of  Metschnikovi,  604 
spirillum  tyrogenum,  603 
Spores  of  moulds   and   fungi  in  atmos- 
pheric dust,  614 

of  non-pathogenic   bacteria,    resist- 
ance of,  to  heat,  158 

Spring  water,  number  of  bacteria  in,  634 
Sputum,    examination    of,    for  bacillus 

tuberculosis,  470 
micrococcus  tetragenus  in,  411 
separation  of  bacilli  from,  by  sedi- 
mentation, 486 

tuberculous,  estimation  of  the  num- 
ber of  bacilli  in,  479 
Stab  cultures,  69 

character  of  growth  in,  70 
of  anaerobic  bacteria,  81 
from  roll-tube  colonies,  77 
Stained  preparations,  the  photographing 

of,  104 

Staining  methods  in  bacteriology,  25 
of  a  dried  bacterial  film,  27 
sections   of    gelatin    stab   cultures, 

36 

bacteria  in  tissues,  34 
sections  of  tuberculous  tissues,  471 
of  spores,  31 

Staining  materials  and  methods  for : 
bacillus  acidiformans,  537 
bacillus      acne     contagiosa     of 

horses,  557 
bacillus  alvei,  556 
bacillus  anthracis,  424 
bacillus  of  Babes  and  Oprescu, 

526 
bacillus  of  Beck,  566 


INDEX. 


703 


Staining  materials  and  methods  for: 

bacillus  of  Belfonti  and  Pasca- 

rola,  508 

bacillus  capsulatus,  521 
bacillus    cavicida    havaniensis, 

516 
bacillus  of  Cazal  and  Vaillard, 

526 
bacillus   of  cholera    in    ducks, 

"503 

bacillus  coli  communis,  580 
bacillus  crassus  sputigenus,  517 
bacillus  cuniculicida  havanien- 
sis, 538,  540 
bacillus  diphtherias,  453 
bacillus  dysenterise,  569 
bacillus  of  Emmerich  and  Wei- 

bel,  566 

bacillus  endocarditidis  capsula- 
tus, 556 
bacillus  endocarditidis  griseus, 

555 

bacillus  enteritidis,  520 
bacillus  erysipelatos  suis,  512 
bacillus  of  Fiocca,  547 
bacillus  of  Friedlander,  396 
bacillus  of  grouse  disease,  520 
bacillus  of  hog  cholera,  504 
bacillus  of  influenza,  463 
bacillus  of  intestinal  diphtheria 

in  rabbits,  Ribbert's  method, 

459 

bacillus  lactis  aerogenes,  536 
bacillus  leporis  lethalis,  541 
bacillus  leprse,  487 
bacillus  of  Lucet,  526 
bacillus  mallei,  489 
bacillus   of   Nicolaier  (capsule), 

560 

bacillus  cedematis  inaligni,  581 
bacillus   cedematis  maligni  No. 

II.,  588 

bacillus  piscicidus,  567 
bacillus     of     purpura     hsemor- 

rhagica  (Babes),  558 
bacillus    of      purpura    hsemor- 

rhagica  (Kolb),  559 
bacillus      of    purpura    hsemor- 

rhagica  (Tizzoni  and  Giovan- 

ninl),  558 


Staining  materials  and  methods  for : 

bacillus    pyocyaneus    pericardi- 

tidis,  547 

bacillus  pyogenes  filiformis,  567 
bacillus  cf  rhinoscleroma,  497 
bacillus  of  septicaemia  hsemor- 

rhagica,  500 

bacillus  solanacearum,  571 
bacillis  of   swine  plague,  Mar- 
seilles, 509 

bacillus  of  syphilis,  495 
bacillus  of  symptomatic  anthrax, 

586 

bacillus  of  tetani,  578 
bacillus  tuberculosis,  30,  469 
bacillus  typhi  abdominalis,  437 
bacillus  typhi  niurium,  524 
bacillus  of  von  Dungern,  562 
diplococcus  intracellularis  men- 

ingitidis,  410 

micrococcus  bothryogenus,  412 
micrococcus  gonorrhoea,  392 
micrococcus  of  Manfredi,  413 
micrococcus  tetragenus,  411 
proteus  hominis  capsulatus,  518 
proteus  lethalis,  554 
proteus  septicus,  554 
proteus  vulgaris,  549 
spirillum  cholerse  asiaticae,  594 
spirillum  of  Finkler  and  Prior, 

602 

spirillum  of  Metschnikovi,  604 
spirillum  of  Obermeieri,  590 
spirillum  tyrogenum,  603 
staphylococcus  pyogenes  aureus, 

374 

streptococcus  pyogenes,  383 
Staphylococcus,  grouping  of,  21 
epidermidis  albus  (Welch),  380 
pyogenes  albus,  380 

albus,    thermal   death-point  of, 

155 

aureus,  373 

aureus,  resistance  of,  to   freez- 
ing, 153 
aureus,  thermal  death-point  of, 

155 

aureus    toxalbumins    from  cul- 
tures of,  150 
citreus,  381 


704 


INDEX. 


Staphylococcus  pyogenes  citreus,  thermal 

death-point  of,  155 
Starvation,  influence  of,  on  development 

of  pus  cocci,  224 
Steam  under  pressure  as  a  disinfectant, 

159,  212,  213 
compared  with  streaming  steam, 

215 

as  a  sterilizer,  56 
sterilizers,  55 

Sterilization  of  media,  etc.,  37,  52 
by  filtration,  58 

imperfect,  errors  arising  from,  62 
of  a  piston  syringe,  97 
of  a  platinum  loop  for  inoculation, 

63 

Sternberg's  bacillus  X,  538 
bulbs,  38,  66 

discovery  of  bacillus  coli  communis 
in  the  blood  and  organs  of  yellow- 
fever  cadavers,  530 
experiments  on  attenuation  of  virus 

by  antiseptic  agents,  131 
on  the  thermal  death-point  of 

bacteria,  154 

method  of  collecting  blood  serum,  38 
liquid  culture  method,  65 
method  of  cultivating  anaerobic  bac- 
teria, 83 

of  determining  the   germicidal 
power  of  chemical  agents  on 
anthrax  spores,  168 
syringe  for  inoculation,  97 
theory  of  immunity,  252 
thermo-regulator,  92 
tubes    for  the  collection    of    water 

samples,  627 
Stomach,  bacteria  of,  not  concerned  in 

digestive  action,  660 
of  nursing  infants,  bacteria  in,  659 
Strauss'  method  of  diagnosticating  glan- 
ders, 493 
Streak  cultures   of  bacillus  septicaemias 

haemorrhagicae,  500,  509 
Strebel's  protective  inoculations,  348,  349 
Streptococci,  17,  129 
grouping  of,  22 
infection  of,  347 

Streptococcus  agalactise  contagiosae,  419 
articulorum  (Loffler),  386 


Streptococcus  bombycis,  415 

coryzse  contagiosae  equorum,  418 
erysipelatos,  382 

action  of  chlorine  on,  177 
lanceolatus  Pasteuri  (Ganieleia),  398 
longus  (von  Lingelsheim),  382 

germicidal   power  of   chemicals 

on,  385 

of  mastitis  in  cows,  417 
mastitis  sporadiae,  420 
perniciosus  psittacorum,  419 
pyogenes,  346,  382 

in  diphtheria,  449,  452,  453,  461 
thermal  death-point  of,  155,  384 
malignus  (Fliigge),  388 
septicus  (Fliigge),  415 
Strict  anaerobics,  16 
parasites,  15,  221 
Sucholoalbumin,  323 
Sucholotoxin,  323 
Sugar  as  a  filter  in  air  analysis,  620 
Sulpho-carbolic   acid,  germicidal  power 

of,  197 

Sulphur  dioxide,   influence  of,  on  bac- 
terial growth,  176 
as  a  disinfectant,  212,  213,  214 
Sulphuretted  hydrogen,  a  bacterial  pro- 
duction, 141 

Sulphuric  acid  as  a  decolorizer  in  stain- 
ing, 28 

action  of,  on  bacterial  growth,  180 
action  of,  on  micrococcus  pneu- 

moniae  crouposaa,  405 
Sulphurous  acid,  action  of,  on  bacterial 

growth,  180 

Sunlight,  importance  of,  as  a  disinfect- 
ant, 162 

Susceptibility  to  disease,  233 
Susotoxin,  323 

of  Novy  from  cultures  of  bacillus  of 

hog  cholera,  505 
Swine  plague,  343 

(American)  bacillus  of  (Salmon 
and    Smith),    identical    with 
bacillus    septicaemias  haemor- 
rhagicae, 499 
practical  measures  for  arresting, 

345 

protective  inoculations  against, 
322 


INDEX. 


705 


Symptomatic  anthrax,  347 

bacillus  of,  585 
Syphilis,  Lustgarten's  bacillus  of,  494 

not  obtained  in  cultures,  496 

TANNIC  acid  as  a  disinfectant,  183 
Tartaric  acid  as  a  disinfectant,  183 
Temperature,    influence   of,  on  bacterial 

growth,  126,  153 
on  spore  formation,  123 
on    the    destruction    of    organ- 
isms, 5 
resistance    of    anthrax  spores  to  a 

high,  427 
animal,  influence  of.  on  immunity, 

235 
Test  tubes,  use  of,  for  cultures,  62 

sterilization  of,  53 
Tetanin,  149 

Brieger's  method  of  obtaining,  352 
Tetanotoxin  of  Brieger,  149,  352 
Tetanus,  a  traumatic  infection,  350 
antitoxin,  260 

curative  power  of,  359 
Tizzoni    and    Cattani 's  experi- 
ments on,  355 
bacillus,  578 

cultures,  toxalbumin  of,  150,  151 
poison  of,  352 

neutralized  by  the  blood  of  an 

immune  animal,  354 
not  neutralized  by  the  blood  of 

chickens,  355 
Tetrads,  22,  120 

Tetraiodphenolphthalein,  antiseptic  val- 
ue of,  179 

Texas  fever  in  cattle,  bacillus  of,  508 
Thallophytes  of  Sachs,  11 
Therapeutic   value  of  antitetanic  blood 

serum,  356 

Thermal  death-point  of  bacteria,  52,  155 
of  bacillus  of  Beck,  566 
of  bacillus  cavicida  havaniensis, 

517 
of  bacillus  of  Cazal  and  Yaillard, 

525 

of  bacillus  coli  communis,  532 
of    bacillis    of    Emmerich    and 

\Veibel,  566 

of  bacillus  erysipelatos  suis,  513 
45 


Thermal  death-point  of  bacillus  of  Laser, 

524 

of  bacillus  piscicidus  agilis,  565 
of  bacillus  pyocyaneus,  543 
of  bacillus  septicaemias  hsemor- 

rhagicae,  501 
of  bacillus  typhi  abdoruinalis, 

441 
of  spirillum  choleras  asiaticse, 

597 

Thermo-regulators,  88 
Thoinot's  method  of  isolating  the  typhoid 

bacillus,  444 

Thyme,  antiseptic  power  of,  203 
Thymic  acid,  action  of,  on  bacteria,  183 
Thymol,  antiseptic  power  of,  207 

action  of,  on  bacillus  tuberculosis, 

478 

Thymus  bouillon,  246,  357 
preparation  of,  359 
immunization  to  tetanus  by  cul- 
tures in,  359 

Tin  chloride  as  an  antiseptic,  195 
Tizzoni  and  Cattani,  on  the  preservation 

of  tetanus  antitoxin,  359 
experiments  favorable  to  Koch's 

tuberculin,  479 
Tizzoni    and    Giovannini's   bacillus   of 

purpura  hsernorrhagica,  558 
Tobacco  smoke,  antiseptic  power  of,  207 
Torula  chains,  22 
Toussaint  on  fowl  cholera,  288 
Toxaemia,  definition  of,  498 

caused  by  anaerobic  bacteria,  578 

by  saprophytic  bacilli,  528,  529 
in  diphtheria,  449 

Toxalbumin  of  diphtheria  cultures,  312 
of  hog  cholera,  505 
of  hydrophobia,  327 
of  tetanus  cultures,  353 
of  typhoid  culture,  150 
Toxalbumins,  146,  149 
Toxic  power  of  the  culture  products  of 

bacillus  tetani,  353 
of  virulent  cultures  neutralized 
by  the  blood  of  immune  ani- 
mals, 259 
Toxin  of  anthrax,  430 

immunity  produced  by,  427 
of  cholera  cultures,  610 


706 


INDEX. 


Toxin  of  bacillus  of  hog  cholera,  505 
of  influenza,  465 
piscicidus  agilis,  565 
tuberculosis,  152,  361 
Toxins,  146,  151 
Toxophylaxin,  265 
Trachoma,  pus  cocci  in,  390 
Tribroniophenol  as  a  disinfectant.  179 
Trikresol,  germicidal  power  of,  202 
Trimethylamine,    a  non-toxic  ptomain, 

147 

Trudeau's  successful  antitubercular  inoc- 
ulations, 366 
Tryptic  enzymes,  135 
Tubercle  bacilli,  method  of  staining  in 

tissues,  36 
in  milk  of  cows,  232 
bacillus  (see  Bacillus  tuberculosis) 
Tuberculin,  action  of,  on  tuberculous  ani- 
mals, 479 

Bujwid's  method  of  preparing,  363 
Helman's  method  of  preparing,  363 
Koch's,  151,  361 
use    of,    by   various  experimenters, 

364,  365 

Tuberculocidin  of  Klebs,  363 
Tuberculosis,  carnivora  immune  to,  233 
among  cattle,  prevalence  of,  482 
usual  methods  of  infection,  481 
protective  inoculations  in,  360 
Tuberose,  antiseptic  power  of,  203 
Turpentine,    antiseptic    power    of,  203, 

206 
Tympanic   cavity,    bacillus   pyocyaneus 

present  in  inflammation  of,  545 
Tyndall's  optically  pure  air,  5 
Typhoid  bacilli  and  colon  bacilli,  varie- 
ties of  the  same  species?  447 
bacillus,  431,  436 

attenuation  of,  242 
biological  characters  of,  438 
culture  medium  for,  47 
dead  animal    matter  a  suitable 

nidus  for,  434 

destroyed  by  gastric  juice,  658 
difficulty  of  differentiating,  440 
influence  of  light  on  the  growth 

of,  161 

morphology.  436 
pathogenesis  of,  442 


Typhoid  bacillus,  pathogenic  to  certain 
of  the  lower  animals,  but  does 
not  convey  typhoid  fever,  436 
pus  production  by,  448 
resistance  of,  to  freezing,  153 
thermal  death-point  of,  155,  441 
vitality  of,    in    various  media, 

442 
vitality  of,  in  moist  atmospheres, 

159 
vitality  of,  when  buried  in  the 

soil,  645 
colonies  in  the  spleen,  post-mortem 

multiplication  of,  434 
cultures,  toxalbumin  of,  150 
fever,  a  disease  of  man,  233 

failure  to  communicate  it  to  ex- 
perimental animals,  435,  442 
heat  as  a  disinfectant  for,  154 
protective  inoculations  in.  3i»7 
second  attacks  of,  243 
Typhotoxin,  148,  222 

of  Brieger,  442 
Tyrotoxicon,  148 

ULCERATIVE  endocarditis,  379,  386 
Underclothing,  worn,  bacteria  on,  649 
Urea,  fermentation  of,  140 
Uric  acid,  influence  of,  on  the  bacterici- 
dal activity  of  blood  serum,  238 
Urine  as  a  culture  medium,  39 

germicidal  power  of,  211 
Uterus,  free  from  bacteria  in  health,  654 

VACCINE  virus,  influence  of  temperature 
on,  156 

Vagina,  bacteria  of,  654 

Vaillard,  immunity  to  tetanus  produced 
in  rabbits,  355 

Valerian,  antiseptic  power  of,  203 

Valerianic  acid  as  a  disinfectant,  183 

Van  Ermengem's  method  of  staining 
flagella,  33 

Veal  broth  peptonized  as  a  culture  me- 
dium for  bacillus  tuberculosis,  477 

Vegetable  infusions  as  culture  media,  41 

Veins,  injections  into,  98 

Venom,  serpent,  immunization  against, 
262 

Verbena,  antiseptic  power  of,  203 


INDEX. 


707 


Vesuvin  as  a  stain,  25 

as  a  double  stain  in  bacterial  prepa- 
rations, 28 
Vibrio,  general  characters,  18 

aquatilis  (Giinther),  remotely  resem- 
bling the  spirillum  of  cholera,  <306 
Berolinensis  (Neisser),  its  differenti- 
ation from  the  cholera  spirillum, 
607 

Danubicus  (Heider),  its  differentia- 
tion from  the  cholera  spirillum, 
608 

of  Ehrenberg,  3,  10 
helcogenes  ^Fischer),  remotely  resem- 
bling the  cholera  spirillum,  607 
Metschnikovi  (Gamele'ia),  604 
Vibrion  septique  (Pasteur),  580 
Vibrionieus  of  Ehrenberg,  3,  10 
Vibrio-proteus,  602 
Vibrios  of  Celli  and  Santori,  60S 

resembling  the  spirillum  of  cholera, 

600 

list  of  species  found  in  water,  639 
phosphorescent,  not  genuine  cholera 

germs,  608,  609 

Vincent's  method  of  detecting  colon  ba- 
cilli in  water  supplies,  640 
Violet    rays    of    spectrum,    germicidal 

power  of,  161 
Virulence,   attenuation  of,   in  cultures, 

130,  388 

intensification  of,  132,  388 
recovery  of,  132 
Viscous  fermentation,  141 
Vital  resistance  theory  of  immunity,  252 
Vitality  of  bacillus  of  hog  cholera,  505 
of  the  typhoid  bacillus  in  cultures, 

442 

Von  Dungern's  capsule  bacillus  distin- 
guished from  others  of  similar  mor- 
phology, 562 

WATER,  action  of,  on  the  cholera  spiril- 
lum, 597 
bacteria  in,  626  <- 

list  of  species  found  in,  638 
number    not  dependent  on   the 
amount  of  organic  matter  pres- 
ent, 635 
distilled,  growth  of  bacteria  in,  635 


Water,  pathogenic  bacteria  that  may  be 

present  in,  636 
bacteria,  detection  of,  640 
bacteriological  analysis  of,  628 
collection  of.  for  analysis,  627 
importance  of  immediate  exam- 
ination, 627 
chemical  analysis  of,  advantages  of, 

631 
filtration,   efficiency  determined   by 

counting  colonies,  641 
supplies,  detection  of  colon   bacilli 

in,  535 
difficulty    of     finding     typhoid 

bacilli  in,  441,  444 
sanitary    inspection    of    source 
more  important  than  bacterio- 
logical or  chemical  analysis, 
641 
vibrios,     remotely    resembling    the 

spirillum  of  cholera,  606 
Waters,  surface,  frequency  of  the  colon 

bacilli  in,  640 
Weichselbaum's  diplococcus   intracellu- 

laris  ineningitidis,  410 
micrococcus  endocarditidis  rugatus, 

416 
Weigert's  method  of  staining  bacteria  in 

tissues,  35 
of  staining  used  in  sections  of 

nodules  of  glanders,  493 
Welch  and  Abbott's  cultivation  of  the 
bacillus  of  diphtheria  on  po- 
tato, 455 
inoculations  of  kittens  with  the 

diphtheria  bacillus,  452 
Well  water,  number  of  bacteria  in,  634 
Widal  reaction  given  by  blood  serum  of 
those  subjected  to  antityphoid 
inoculation,  369 
in  Malta  fever,  421 
Wildseuche,  287,  499 
Winogradsky's  silicate  jelly,  47 
Wintergreen,  antiseptic  power  of,  203 
Wolffhugel's  experiments  on  the  thermal 

death-point  of  bacteria,  154 
Wolter's  opinion  that  positive  results  in 
attempts  to  inoculate  leprosy  were  due 
to  associated  tubercle  bacilli,  488 
Woollen  underclothing   bacteria  on,  649 


708 


INDEX. 


Wool-sorter's  disease,  422,  430 

a  pulmonary  affection,  2."H 
Woven  underclothing,  bacteria  on,  649 
Wurtz's  method  of  identifying  typhoid 
bacilli  in  water  supplies,  44-~> 

XYLOL  for  solution  of  Canada  balsam,  28 

YELLOW  fever,  insusceptibility  of  negro 

to,  234 

cadavers,  bacillus  coli  conmmnis 
in  the  blood  and  organs  of,  530 
milk.  668 

Yersin's    attenuated  varieties  of  diph- 
theria bacillus,  oOD 
description  of  the  bacillus  pestis,  563 

ZEA  mays,  bacterial  diseases  of,  575 


Zedvary,  antiseptic  power  of,  203 
"Zeitsckrift  fur  Hygiene,"  8 
Ziehl's  carbol-fuchsin  solution.  2!) 
for  bacillus  lepra,  487 
for   bacillus   tuberculosis,   469- 

471 

for  the  typhoid  bacillus,  438 
Ziehl-Nelson  method  of  staining  the  tu- 
bercle bacillus,  30,  36 
Zinc  chloride  as  an  antiseptic  and  disin- 
fectant, 195,  212 
sulphate  as  a  germicide,  196 
Zooglea,  21 

formed  by  bacillus  solanacearum,  571 

proteus  mirabilis,  553 
Zopf,  classification  of,  12 
Zymogenic  organisms,  14 


